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Accepted Manuscript Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Saireudee Chaturantabut, Arkadi Shwartz, Kimberley J. Evason, Andrew G. Cox, Kyle Labella, Arnout G. Schepers, Song Yang, Marianna Aravena, Yariv Houvras, Liliana Mancio-Silva, Shannon Romano, Daniel A. Gorelick, David E. Cohen, Leonard I. Zon, Sangeeta N. Bhatia, Trista E. North, Wolfram Goessling PII: S0016-5085(19)30031-9 DOI: https://doi.org/10.1053/j.gastro.2019.01.010 Reference: YGAST 62366 To appear in: Gastroenterology Accepted Date: 7 January 2019 Please cite this article as: Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG, Yang S, Aravena M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, Bhatia SN, North TE, Goessling W, Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes, Gastroenterology (2019), doi: https://doi.org/10.1053/j.gastro.2019.01.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Page 1: Estrogen Activation of G protein-coupled Estrogen Receptor 1 …€¦ · Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR

Accepted Manuscript

Estrogen Activation of G protein-coupled Estrogen Receptor 1 RegulatesPhosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth inZebrafish and Proliferation of Human Hepatocytes

Saireudee Chaturantabut, Arkadi Shwartz, Kimberley J. Evason, Andrew G. Cox,Kyle Labella, Arnout G. Schepers, Song Yang, Marianna Aravena, Yariv Houvras,Liliana Mancio-Silva, Shannon Romano, Daniel A. Gorelick, David E. Cohen,Leonard I. Zon, Sangeeta N. Bhatia, Trista E. North, Wolfram Goessling

PII: S0016-5085(19)30031-9DOI: https://doi.org/10.1053/j.gastro.2019.01.010Reference: YGAST 62366

To appear in: GastroenterologyAccepted Date: 7 January 2019

Please cite this article as: Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG,Yang S, Aravena M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, BhatiaSN, North TE, Goessling W, Estrogen Activation of G protein-coupled Estrogen Receptor 1 RegulatesPhosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferationof Human Hepatocytes, Gastroenterology (2019), doi: https://doi.org/10.1053/j.gastro.2019.01.010.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.

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GPER1 mTOR

PI3K-AKT

GrowthRegenerationCancer

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Title: Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Short Title: GPER1 enhances liver growth Authors: Saireudee Chaturantabut1, Arkadi Shwartz1, Kimberley J. Evason2, Andrew G. Cox1,3, Kyle Labella1, Arnout G. Schepers4, Song Yang5, Marianna Aravena6, Yariv Houvras7, Liliana Mancio-Silva4, Shannon Romano8, Daniel A. Gorelick8, David E. Cohen6, Leonard I. Zon5,9,10,11, Sangeeta N. Bhatia4,12,13, Trista E. North5,10, Wolfram Goessling1,10,11,12,13,14. 1Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. 2Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA 3Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.. 4Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. 5Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA. 6Division of Gastroenterology & Hepatology, Weill Cornell Medical College, New York, NY 10021, USA. 7Departments of Surgery and Medicine, Weill Cornell Medical College, New York, New York 10021, USA. 8Department of Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL 35293, USA. 9 Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MA 20815, USA. 10Harvard Stem Cell Institute, Cambridge, MA 02138, USA. 11Dana-Farber Cancer Institute, Boston, MA 02215, USA. 12Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, 02139, USA. 13Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 14Divison of Gastroenterology, Massachusetts General Hospital, Boston, MA 02114 Grant Support: This study is supported by NIH NCI 5K08CA172288 (K.J.E.), R24OD017870 (L.I.Z.; W.G), R01DK090311, R01DK105198 (W.G.) and The Claudia Adams Barr Program in Cancer Research (W.G.). W.G. is a Pew Scholar in the Biomedical Sciences. Abbreviations: ANAS, anastrozole; ANT, adjacent normal tissue; Akt, protein kinase B; BPA, bisphenol A; BrdU, bromodeoxyuridine; CC, cholangiocarcinoma; DAPI, 4′,6-diamidino-2-phenylindole; DMBA, dimethylbenzanthracene; DMSO, dimethysulfoxide; EdU, ethynyldeoxyuridine; ERE, estrogen response element; ERK, extracellular signal-regulated kinases; E2, 17β-estradiol; ESR, estrogen receptor; FACS, fluorescence-activated cell sorting; ful, fulvestrant; GO, Gene Ontology; GPER1, G protein-coupled estrogen receptor 1; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; HNF4α, hepatocyte nuclear factor 4α; hpf, hours post fertilization; hpT, hours post treatment; IACUC - Institutional Animal Care and Use Committee; IHC, immunohistochemistry; ISH, in situ hybridization; mTOR, mechanistic target of rapamycin; MO, morpholino antisense oligonucleotide; Mtz, metronidazole; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TALEN, transcription activator-like effector

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nuclease; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; WT, wild-type. Correspondence: Address correspondence to Wolfram Goessling ([email protected]), Genetics Division, Brigham and Women’s Hospital, NRB458, 77 Ave Louis Pasteur, Boston, MA 02115, U.S.A, phone: 617-525-7710, fax: 617-525-4751. Disclosure: The authors have no conflict of interest to disclose. Transcript Profiling: Accession number in GEO is GSE92544.

Author Contributions: S.C. and W.G. conceived the study, designed experiments, and analyzed data. S.C. conducted all zebrafish experiments. A.S. performed confocal microscopy and Western Blots. K.J.E. acquired human tissues and performed pathological analysis of zebrafish and human liver tumors. A.G.C. analyzed metabolomics data. K.L. performed mRNA rescue, xenoestrogens, and long-term anastrozole zebrafish experiments. A.G.S. and L.M-S. performed primary human hepatocyte experiments. S.Y. and Y.H. analyzed RNA sequencing data. M.A. carried out liver cancer cell line experiments. S.R., D.A.G., D.E.C., L.I.Z., S.N.B, and T.E.N. provided overall input. S.C., T.E.N and W.G. wrote the manuscript. All authors reviewed and edited the manuscript. The authors thank Isaac Oderberg from the Goessling lab for creating the visual abstract, John Asara at the Metabolomics Core at the Beth Israel Deaconess Medical Center for assistance with the metabolomics experiments, and staff of Beth Israel Deaconess zebrafish core facility for fish husbandry.

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Title: Estrogen Activation of G Protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Abstract: Background & Aims: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Methods: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages; liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a co-culture system. We measured levels of G protein-coupled estrogen receptor (GPER1) in HCC and non-tumor liver tissues from 68 patients by immunohistochemistry. Results: Exposure to 17 beta-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the PI3K–Akt–mTOR pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1, compared with non-tumor tissues; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. Conclusions: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. KEY WORDS: sex hormone, signal transduction, transcription regulation, hepatocarcinogenesis

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Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality

worldwide, and the fastest-growing cause of cancer deaths in the U.S.1 Clinically relevant

biomarkers and therapies to detect and prevent HCC do not exist. Interestingly, chronic

liver disease and liver cancer are much more frequent in males,2 and men with cirrhosis

and HCC have elevated serum estrogen levels.3, 4 Further, patients undergoing surgical

liver resection display elevated serum levels of estrogen, suggesting the importance of

estrogenic regulation during liver regeneration.5 The mechanisms by which the liver

senses and responds to estrogen to impact liver growth and cancer formation remain

undetermined.

17β-estradiol (E2) is the most abundant biologically active form of estrogen. Canonical

estrogen signaling is mediated through nuclear hormone estrogen receptors 1

(ESR1/ERα) and 2 (ESR2/ERβ), resulting in transcriptional target gene activation. In

addition, E2 can exert non-canonical activity through the G protein-coupled estrogen

receptor 1 (GPER1, also known as GPR30). While roles of ESR1 and ESR2 have been

widely studied in the context of reproductive biology and cancer,6 the functional

consequences of GPER1 signaling are less well understood. Originally discovered in

breast cancer cells,7 GPER1 regulates proliferation and relaxation of vascular smooth

muscle.8 Several secondary messenger signals have been identified in different cellular

contexts, including Erk,7 PI3K,9 and Ca2+ release.10 GPER1 expression in hepatocytes,

and its potential role in hepatocyte proliferation and organ growth during development,

liver regeneration or cancer progression have not been previously characterized.

Here, we identify the essential function of E2 and GPER1 in the regulation of liver growth.

E2 induces cell cycle progression and increases hepatocyte proliferation and liver size in

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larval zebrafish. Surprisingly, these effects are not mediated through classic nuclear

hormone estrogen receptors, but via GPER1 and downstream activation of PI3K-mTOR

signaling. Importantly, GPER1 promotes sex-specific adult liver growth, and together with

mTOR is required for optimal liver regrowth after injury. In addition, GPER1 directly

modulates liver cancer formation: gper1-/- fish develop significantly fewer and smaller liver

tumors than wild-type siblings. The role of GPER1 is conserved in human liver, as liver

cancer tissue expresses increased GPER1, and primary human hepatocytes activate

AKT/mTOR and proliferate in response to E2. Chemical inhibition of GPER1 in vivo

significantly diminishes E2-induced tumor progression after chemical carcinogenesis,

particularly in males. We propose that GPER1 senses E2 to regulate PI3K/mTOR activity

and cellular proliferation during hepatic development and repair, and as such is an

important therapeutic target for liver cancer prevention and treatment.

Materials and Methods

Zebrafish husbandry

Zebrafish were maintained according to Institutional Animal Care and Use Committee

guidelines (IACUC) at Harvard Medical School.

Chemical Exposures

Zebrafish larvae were exposed to chemical modifiers, dissolved in 0.1% (v/v)

dimethylsulfoxide (DMSO), for 5hrs from 110-115 hours post fertilization (hpf) and

analyzed at 120hpf (unless otherwise specified).11 Adults were treated for 5-7hrs per

exposure. Chemicals are listed in Supplementary Table S5.

Morpholino Injection

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Morpholino oligonucleotides (MO) designed against esr1, esr2a, esr2b,11 gper1, and

mtor (GeneTools, Supplementary Table S3) and mismatched controls were injected into

one-cell stage embryos.

mRNA injection

Human GPER1 cDNA-containing plasmid (HsCD0032896) was transcribed using

mMESSAGE mMACHINE (Ambion). mRNA (200µg) was injected into 1-cell stage

embryos.

Generation of gper1 Mutants

TALENs targeting gper1 were obtained from Addgene (TAL3272, TAL3273).12 Adult

TALEN-injected fish (F0) were out-crossed to WT siblings, and progeny (F1) screened for

somatic mutations by Sanger sequencing (Supplementary Table S1). F1 mutants were

out-crossed for at least 4 generations to avoid possible off-target effects.

Western Blot Analysis

Pooled larvae (n=30-40) and cultured cells were processed for Western blot as previously

described.13 Antibodies are listed in Supplementary Table S4.

Whole Mount in situ Hybridization

Larvae were fixed and ISH performed according to standard protocols.14 gper1 probe was

generously gifted by David Volz.15

Liver Size Analysis

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ISH images were obtained by brightfield microscopy, and fabp10a:GFP reporter larvae

were imaged using fluorescence microscopy. 3D imaging of fabp10a:GFP fish was

performed with a Yokogawa W1 spinning disk confocal microscope. Image quantification

was achieved using ImageJ and Imaris software.

Histology and immunohistochemistry of zebrafish and human tissue

Zebrafish were fixed, sectioned and processed with hematoxylin/eosin (H&E) stains as

described16. For cell size analysis, zebrafish sections were stained with Pan-Cadherin

and 4′,6-diamidino-2-phenylindole (DAPI). Individual cell membrane staining was traced

and the cell area was measured using ImageJ. De-identified formalin-fixed paraffin-

embedded human liver sections were obtained at the University of Utah (IRB 00091019,

clinical characteristics summarized in Supplementary Table S6 and S7) or from

commercially available tissue arrays (OD-CT-DgLiv01-003, US Biomax, Supplementary

Table S7). IHC (antibodies in Supplementary Table S4) and terminal deoxynucleotidyl

transferase dUTP nick-end labeling (TUNEL) staining were performed using established

protocols.14 GPER1 staining was scored by a pathologist (K.J.E.).

Flow Cytometry and Cell Cycle Analysis

Fluorescence-activated cell sorting (FACS) on fabp10a:GFP larvae was performed as

previously described.14 Cell cycle analysis was achieved using the BrdU Flow kit (BD

Bioscience) and incubation in propidium iodide (PI) followed by flow cytometry analysis,

performed with FlowJo software.

qRT-PCR Analysis

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RNA was isolated from zebrafish larvae, processed and analyzed by qRT-PCR as

previously described.17 Primers are listed in Supplementary Table S2.

Chemical Carcinogenesis

Age-matched gper1-/- or wild-type fish were exposed to 5ppm dimethylbenzanthrazene

(DMBA) for 24hrs at 3, 4, and 5 weeks.18 Chemical exposures commenced at 6 weeks (3

times/week). Fish were monitored daily for survival and tumor formation until 33 weeks

post DMBA treatment. Fish were sacrificed upon reaching IACUC-approved tumor limits

or at 33 weeks post DMBA exposure. Histologic analysis was performed in a blinded

fashion by a board-certified pathologist with expertise in zebrafish liver histology (K.J.E.).

Steady-state Metabolomics Analysis

Adult livers were surgically removed and subjected to methanol extraction as previously

described.17, 19 Polar metabolites were identified using LC-MS/MS. Metabolic pathway

analysis was performed with MetaboAnalyst (http://www.metaboanalyst.ca).

Transcriptomic Analysis

RNA was extracted from resected livers, processed and analyzed as previously

described.17 Gene Ontology (GO) terms were analyzed using DAVID (NIAID).

Results

Estrogen enhances liver growth

To demonstrate the potential impact of endogenous estrogen levels on liver size, adult

zebrafish livers were assessed in females and males. Livers of adult female zebrafish

were bigger than that of male siblings, as visualized in hepatocyte reporter fish

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expressing GFP regulated by the fatty acid binding protein 10a promoter,

Tg(fabp10a:GFP) (Figure 1A). Baseline liver:body weight ratios in 10 WT males and

females at 7 months, revealed a significant 2.6-fold difference (Figure 1B). Since

estrogen is one of the major sex hormones determining physiological differences between

males and femails, the potential impact of estrogen on liver growth was examined:

zebrafish were exposed to E2 (10µM) or DMSO daily for six weeks, and liver size and

weight were assessed in the context of total body length and mass (Figure 1A,B). Male

livers responded more significantly to E2 with a 4.8-fold increase in liver:body weight

ratio, while females exhibited a 1.6-fold enhancement (Figure 1B). These results suggest

that liver mass is directly responsive to estrogen.

To uncover the signals by which E2 affects liver size, liver transcriptome analysis was

performed with and without E2. Gene expression in male and female livers was different,

with a subset of genes identified as “female-associated”, indicating genes up-regulated

exclusively in females (Supplementary Figure S1A). These differences, however, are

not solely due to estrogenic effects, as E2 did not simply convert the male liver

transcriptome into one resembling a female: in fact, E2 exposure altered transcriptional

profiles in both sexes (Supplementary Figure S1A). GO analysis revealed that E2

enhanced cell cycle and DNA metabolism-related gene expression (46 genes, p=3.4x10-

5), particularly in males (Figure 1C, Supplementary Figure S1B,C; 324 genes,

p=7.4x10-13). Given that the liver is a primary metabolic organ, we examined whether the

sex and/or E2-associated increase in liver mass correlated with metabolic alterations,

employing steady-state metabolomics profiling (Figure 1D, Supplementary Figure

S1D,E). Metabolite profiles were significantly different between males and females, and

further altered by E2 exposure: Metabolite set enrichment analysis revealed E2 exposure

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altered pyrimidine and purine metabolism in male livers, which is essential for DNA

synthesis and cell proliferation (Figure 1D; 55 metabolites; pyrimidine, p=1.9x10-8; purine,

p=3.5x10-3).17 E2 altered metabolites involved in arginine/proline metabolism and RNA

transcription in both male and female livers. Together, these findings indicate that E2

predominantly affects transcriptional programs and metabolites involved in cell cycling

and proliferation.

To directly investigate the effect of E2 on hepatocytes, without the influence of pre-

existing sex differences, zebrafish larvae were examined before sexual dimorphic

features have developed. Larvae were exposed to E2 for 5hr intervals after hepatocytes

were fully differentiated (>95hpf). E2 increased liver size at 120hpf as analyzed by ISH for

fabp10a; this effect was confirmed by fluorescent imaging of hepatocyte reporters

(Tg(fabp10a:GFP)) and qRT-PCR (Figure 1E,F; p<0.01). E2 also enhanced liver volume

by confocal microscopy (Figure 1G; p<0.01), and total hepatocyte number, as quantified

by FACS (Figure 1H; p<0.0001). To determine the impact of endogenous E2, larvae

were exposed to the aromatase inhibitor anastrozole (ANAS), revealing a decrease in

liver size (Figure 1I,J). Critically, liver size could completely be rescued by escalating

doses of exogenous E2 (Figure 1J; p<0.01). These results indicate that during larval

stages E2 regulates hepatocyte number and liver growth.

Estrogen increases liver size via activation of GPER1

To identify which receptor mediates the effect of E2 on liver growth, larvae were exposed

to selective chemical antagonists for each estrogen receptor, MPP (ESR1),20 PHTPP

(ESR2),21 and G-15 (GPER1),22 alone and with E2. The E2-induced increase in liver size

was specifically inhibited by co-exposure with G-15, but not by ESR1 or ESR2 blockade

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(Figure 2A,B). Selective GPER1 activation with G-1 increased liver size and hepatocyte

number, similarly to E2, while GPER1 inhibition with G-15 reduced liver size

(Supplementary Figure S2A,B). Furthermore, knockdown of gper1, but not nuclear

estrogen receptors esr1, esr2a, esr2b, or esr2a+esr2b, blocked the effect of E2 on liver

growth, confirming specificity of the chemical modifiers (Supplementary Figure S2C).

Importantly, human GPER1 mRNA partially rescued the small liver phenotype in gper1

morphants, demonstrating specificity and evolutionary conservation (Figure 2C,D).

Similarly, GPER1 mRNA injection increased liver size in WT embryos, but not after

exposure to ANAS, revealing the comcomitant importance of endogenous E2 for GPER1

activation (Supplementary Figure 2D). Finally, known environmental GPER1 agonists,

bisphenol A (BPA) and tamoxifen, likewise augmented liver size (Supplementary Figure

2E,F). To confirm that hepatic nuclear hormone receptor-mediated signaling was not

significantly activated following E2 exposure, estrogen response element (ERE) reporter

fish Tg(5xERE:GFP) were imaged to reflect DNA binding by nuclear hormone receptors.23

Fluorescence imaging at 120hpf revealed that short exposure to E2 (110-115hpf) had a

moderate effect on baseline hepatic Esr signaling, while prolonged exposure for 24hrs

significantly increased hepatic ERE activity (Supplementary Figure S2G,H), consistent

with the kinetics of non-genomic signaling versus genomic signaling through

transcriptional regulation. Intriguingly, gper1 expression is dynamically regulated during

development, and strongly localized to the liver at 72-120hpf as determined by ISH and

RT-PCR (Supplementary Figure S3A-C). Together, these data indicate GPER1 impacts

E2-mediated liver growth during development.

To define the role of GPER1 in mediating E2-associated effects on liver growth, gper1

mutant zebrafish were generated using TALENs: a 29-base pair deletion in exon one

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resulted in a premature stop codon, disrupting GPER1 protein production

(Supplementary Figure S3D,E). GPER1 loss, assessed in the Tg(fabp10a:GFP)

reporter background, impaired liver growth substantially after 96hpf, but not earlier,

supporting a requirement for E2 and GPER1 in liver outgrowth (Figure 2E,F). Consistent

with these findings, expression of hepatic progenitor markers, foxA3 and prox1, was not

affected in gper1-/- larvae at 48 or 72hpf (Supplementary Figure S3F,G). Significantly, in

contrast to WT livers, gper1-/- mutants did not respond to E2 or G-1 (Figure 2G,H),

definitively indicating that E2 signals via GPER1 to increase liver size.

Estrogen activates GPER1 to promote cellular proliferation and cell cycle

progression

To determine the cellular mechanism by which E2 enlarges liver size, cell cycle analysis

was performed on propidium iodide-stained fabp10a:GFP+ hepatocytes at 120hpf (Figure

3A). E2 and G-1 significantly increased hepatocytes in S and G2/M phase (S:21.8%;

G2/M:9.4%) compared to controls (S:14.9%; G2/M:4.2%; p<0.01); importantly, this effect

was blocked by G-15 (Figure 3B; S:16.2%; G2/M:2.9%; p<0.01. In contrast, in gper1-/-

mutants, whole-larvae cell cycle analysis at 120hpf revealed impaired cell cycle

progression (S:14.8%; G2/M:8.1%; p<0.01) compared to age-matched controls (Figure

3C,D, S:23.3%; G2/M:8.5%). In WT embryos, cellular proliferation, marked by BrdU

incorporation (Supplementary Figure S4A,B) and PCNA staining, was enhanced after

E2 or G-1 exposure (Figure 3E,F, Supplementary Figure S4D,E). This impact was not

sustained, however, as 45hrs after E2 exposure, cell cycle parameters and BrdU

incorporation were comparable to controls (Supplementary Figure S4C,D,E). In

contrast, G-15 decreased PCNA+ hepatocytes in WT embryos and inhibited E2-induced

increases. Any impact of E2 on hepatocyte viability was excluded by TUNEL staining

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(Figure 3E, Supplementary Figure S4F). Cell size alterations contributing to organ size

were examined by pan-Cadherin immunostaining, revealing a significant 50% increase in

hepatocyte area upon E2 and G-1 exposure (Figure 3G,H). These results indicate that

E2 promotes hepatocyte proliferation, cell cycle progression and overall size via GPER1,

leading to increased liver mass.

Estrogen signals through GPER1 to stimulate the PI3K-Akt pathway

To determine the downstream signals by which GPER1 enhances liver growth, a targeted

approach was employed, based on prior work indicating many G protein-coupled

receptors activate PI3K signaling. 24 To define a potential interaction between GPER1

and PI3K-Akt signaling, larvae were exposed to the PI3K inhibitor LY292002, PI3K

activator 740Y-P, and Akt inhibitor MK-2206. Co-exposure with either LY292002 or MK-

2206 diminished E2-induced liver growth (Figure 4A,B; Supplementary Figure S5A).

MK-2206 also decreased liver size in WT and gper1-/- mutants, suggesting that

E2/GPER1-PI3K/Akt signaling is essential for larval liver outgrowth. In contrast, activation

of PI3K signaling by 740Y-P increased liver size in WT larvae and rescued gper1

morphants or mutants (Figure 4A,B; Supplementary Figure S5B). Cell proliferation as

measured by PCNA was decreased by MK-2206 and increased with 740Y-P, respectively

(Supplementary Figure S5C,D). As PI3K can activate both Akt and MAPK-Erk

pathways, MAPK-Erk signaling was also examined. Western blot for phosphorylated Akt

(p-Akt) and phosphorylated Erk (p-Erk) showed that E2 increased p-Akt with minimal

effects on p-Erk (Figure 4C). gper1-/- mutants exhibited decreased baseline p-Akt levels

compared to WT, which was restored by 740Y-P exposure. These data demonstrate that

E2-GPER1 activates the PI3K-Akt pathway to increase liver growth.

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Estrogen activates mTORC1 pathway via GPER1 to increase liver size

To determine the effector of E2-GPER1-mediated activation of PI3K-Akt signaling, the

role of the mechanistic target of rapamycin complex 1 (mTORC1) was investigated. Co-

exposure with the mTORC1 inhibitor rapamycin diminished the enlarged liver induced by

E2 or G-1 (Supplementary Figure S5E,F). Similarly, mtor knockdown decreased liver

size and blocked the effect of E2 (Supplementary Figure S5G,H). Hypomorphic mtor-/-

mutants25 exhibited smaller livers that did not respond to E2 or G-1 (Figure 4D,E),

indicating that GPER1 acts upstream of mTORC1. Finally, Western blot of mTORC1

target ribosomal protein S6 (S6) revealed increased phosphorylation with E2 and G-1

(Figure 4F); importantly, IHC staining localized increased p-S6 to the liver

(Supplementary Figure S5I). These effects were abolished in gper1-/- and mtor-/-

mutants (Figure 4F). Together, these functional and biochemical analyses demonstrate

that E2-GPER1-dependent regulation of liver size is mediated by activation of PI3K-

mTORC1.

Estrogen promotes liver regeneration via activation of GPER1 and mTORC1

pathways

Our previous work indicated strong conservation of developmental regulatory signals in

liver regeneration.14,13 To determine whether the GPER1-mTOR pathway impacts liver

repair, a genetic ablation strategy was utilized: expression of bacterial nitroreductase in

hepatocytes (Tg(fabp10a:CFP-NTR)) enables targeted ablation upon metronidazole (Mtz)

exposure.26 Larvae were exposed to Mtz from 84-120hpf followed by chemical incubation

and liver assessment at 30hrs post treatment (30hpT; Figure 5A). E2 or G-1-exposed

larvae demonstrated significantly more liver regrowth compared to controls (Figure 5B,C;

p<0.01, p<0.0001). In contrast, gper1-/- mutants exhibited significantly less regrowth

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(p<0.0001), which was not enhanced by E2 or G-1 (Figure 5B,C). These results indicate

that E2-GPER1 signaling promotes liver growth after injury.

To confirm the role of the E2/GPER1-PI3K/mTOR axis during liver repair, compound

mtor-/-;fabp10a:CFP-NTR fish were examined. Liver regrowth in mtor-/- mutants was

significantly reduced compared to controls (p<0.0001) and not enhanced by E2 or G-1

exposure (Figure 5B,C), highlighting a general requirement for mTOR during liver

regeneration and its requirement for mediating the effect of E2. IHC demonstrated lower

p-S6 levels in livers of both gper1-/- and mtor-/- mutants after injury, indicating a failure to

upregulate mTORC1 signaling upon hepatocyte ablation (Supplementary Figure 6A,B).

Collectively, our data reveal a direct connection between E2 and mTORC1 in promoting

liver growth during regeneration.

GPER1 mediates adult liver growth

Since E2-GPER1 promotes liver growth and repair in zebrafish larvae, it may influence

adult liver biology as implied in our initial observations (Figure 1). Steady-state liver

weight and liver:body weight ratios in WT and gper1-/- males were not significantly

different (Figure 6A,B; Supplementary Figure S6C); in contrast, E2 induced an

elevation in male liver weight that was significantly reduced in gper1-/- mutants (Figure

6A,B), indicating sensitivity to exogenous E2 that may be contributed to by a 6-fold

increase in gper1 expression in male livers over females (Figure 6C). In contrast, loss of

gper1 in females reduced liver weight and liver:body weight ratios (Figure 6A,B); and

inhibition of endogenous estrogen production by anastrozole also reduced female

liver:body weight ratios in WT, but not in gper1-/- mutants (Supplementary Figure

S6F,G). In addition, body weight and length measurements revealed a systemic effect of

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GPER1 loss in both sexes, consistent with the importance of mTOR for metabolic

homeostasis (Supplementary Figure S6D,E). Further, comparision of all parameters

between WT and gper1-/- animals also suggests an impact of E2 on liver weight and

whole body weight and length independent of GPER1 signaling, likely through ESR

activity. Increased levels of p-Akt after 5 hours of E2 exposure in WT male livers, but not

those of gper1-/- mutants (Figure 6D) further highlight the persistence of E2-GPER1-

PI3K-Akt regulatory axis in adults.

GPER1 promotes human hepatocyte growth

To demonstrate that the pro-proliferative effects of E2 had relevance to human

physiology, a human primary hepatocyte co-culture system was employed.27 E2

increased proliferation in male hepatocytes, doubly labeled with hepatocyte nuclear factor

4 alpha (HNF4α) and ethynyldeoxyuridine (EdU), in a concentration-dependent manner

(Figure 6E,F; p<0.01). Further, E2 exposure enhanced p-AKT and p-S6 levels (Figure

6G,H,I). Importantly, GPER1 is expressed in human hepatocytes, but not in the co-

cultured support cells (Supplementary Figure S6H), indicating a cell-autonomous effect

of E2/GPER1 on hepatocyte proliferation. In human HepG2 cells, E2-associated

increases in p-AKT and p-S6 were blocked by G-15 co-exposure (Supplementary

Figure S7A), highlighting that E2 similarly signals via the Akt-mTOR pathway in human

liver cancer. Together, these results demonstrate the conserved role of E2-GPER1-PI3K-

Akt-mTOR in promoting human hepatocyte proliferation.

GPER1 expression marks human cirrhotic liver and HCC

To assess the potential clinical relevance of GPER1 activity in human liver, GPER1

expression was quantified in sample from 68 individual patients: 30% of non-cirrhotic

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livers exhibited patchy-to-diffuse GPER1 staining (Figure 7A,B; Supplementary Figure

S7B, Supplementary Table S6, Supplementary Table S7, staining score ≥1+). GPER1

is expressed at higher levels in normal male livers than females, consistent with the

zebrafish analysis (Supplementary Figure S7B,C). In contrast, 91% of cirrhotic livers,

and 86% of HCC samples exhibited positive patchy-to-diffuse cytoplasmic GPER1

staining, with enhanced reactivity of hepatocytes around portal tracts and fibrous bands

(Figure 7A,B; non-cirrhotic vs. cirrhotic, p<0.05). These results indicate that GPER1 is

increasingly expressed in cirrhotic livers and HCC, implying a potential role for GPER1 in

human hepatocarcinogenesis.

GPER1 contributes to sex-dimorphism in cancer formation and progression

Dysregulation of signaling pathways controlling embryonic development and adult tissue

homeostasis are frequently causative in carcinogenesis.28, 29 Given the role of E2-GPER1

in liver growth and its elevation in human liver cancer samples, its impact on cancer

formation was examined in a well-established model of chemical liver carcinogenesis

employing DMBA:18 gper1-/- mutants exhibited a significant 50% reduction in liver cancer

incidence compared to DMBA-treated controls (Figure 7C; 42.8%, n=21 vs. 22.2%, n=45;

p<0.05): tumor number per liver as a measure of tumor initiation in the entire cohort was

decreased in gper1-/- mutants (Figure 7D; p<0.05). Moreover, average liver tumor size,

as an indicator of tumor progression, was decreased by >70% in gper1-/- mutants (Figure

7E; 0.7 vs. 0.2 mm, p<0.01). These results imply that GPER1 function directly influences

both the initiation and progression of liver cancer, suggesting a possible therapeutic role

for receptor blockade.

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To determine if E2-GPER1 is a possible therapeutic target in liver carcinogenesis, DMBA-

exposed fish were subsequently treated three times weekly with DMSO, E2, or E2+G-15

(Figure 7F). Significantly, E2-exposed fish had decreased survival compared to controls

and those concomitantly exposed to E2+G-15 (Figure 7G; p<0.0001). E2 exposure

almost doubled liver tumor growth at 33 weeks post carcinogenesis, as indicated by

larger overall tumor size (Supplementary Figure S7D; mean 0.5 vs. 0.9 mm, p<0.01).

Strikingly, G-15 co-exposure reduced the E2-associated increase in tumor burden to

below baseline levels (0.9 vs. 0.4 mm, p=0.01). Importantly, the ESR antagonist

fulvestrant (Ful) in combination with E2 did not decrease tumor size (Figure 7H,

Supplementary Figure S7D), but rather caused an increase in tumor size in both sexes.

Consistent with the known agonistic potential of selective ESR inhibitors on GPER1,30

fulvestrant exposure increased total and phosphorylated Akt in zebrafish larvae

(Supplementary Figure S7E). Histological analysis revealed the presence of

hepatocellular adenoma (HCA), hepatocellular carcinoma (HCC) and cholangiocarcinoma

(CC) without the chemical treatments affecting overall tumor spectrum (Supplementary

Figure S7F,G). Sex-stratified analysis revealed that E2 effectively tripled tumor size in

males (Figure 7H; p<0.001, Supplementary Figure S7D); likewise, blockade by G-15

predominantly reduced tumor size in males (p<0.05), with less impact in female fish.

Fulvestrant enhanced tumor size particularly in males (p<0.001). Together, these results

indicate that GPER1 is an essential component of the sexually dimorphic response to E2

during liver growth, including carcinogenesis, which may have therapeutic relevance for

the detection and treatment of human liver disease.

Discussion

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Liver cancer is the second most common cause of cancer death worldwide, and

predominantly affects men.2 Here, we identified a novel role for GPER1 in hepatocytes

that cell-autonomously regulates larval liver growth and contributes to sex-dimorphisms

observed in liver cancer incidence and growth. E2 promotes hepatocyte cell cycle

progression and proliferation through GPER1 and downstream activation of PI3K-mTOR

signaling. Importantly, the GPER1-mTOR pathway remains essential for liver repair.

Prospective in vivo longitudinal carcinogenesis assays identify GPER1 as an important

factor promoting E2-mediated liver cancer initiation and progression. Sex stratification

analysis demonstrates a male-predominant effect. Importantly, while ESR inhibition

worsens outcome, pre-clinical studies utilizing a selective GPER1 antagonist highlight the

therapeutic potential of GPER1 blockade for liver cancer treatment. Likewise, increased

GPER1 expression in human cirrhotic livers and HCC indicate its possible utility as a

biomarker for disease progression.

The mTOR pathway is essential for organ size regulation,31 functioning as a growth

checkpoint that tightly coordinates cell growth with environmental cues, such as cellular

stress, energy status, and amino acid availability.29 Here, we reveal a previously

unidentified role of E2-GPER1 acting upstream of mTORC1 to promote hepatocyte

proliferation and liver growth. We further identify the impact of E2-GPER1 activation on

pyrimidine and purine metabolism, supporting recent work that mTORC1 is essential for

de novo pyrimidine and purine biosynthesis to meet the anabolic demands of rapidly

proliferating cells.32, 33 Prior studies have reported sexually dimorphic mTOR activity in the

liver.34, 35 Our study is the first to elucidate how hepatic mTOR may sense a sex-specific

environment through GPER1 and influence liver growth.

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Several clinical and laboratory studies have shown sexually dimorphic responses after

hepatectomy, with females being more tolerant and having a higher rate of liver

regeneration.36 E2 levels reportedly increase after liver injury,37 and improve impaired

liver regeneration in ovariectomized female mice.38 The mechanisms involved, however,

had been incompletely understood. Here, we reveal that E2 specifically activates GPER1

and mTORC1 to promote liver regrowth after injury. The importance of E2 can be gleaned

from clinical observations: E2 levels rapidly increase after liver resection in patients.5

Further, pregnancy is associated with hepatomegaly,39 and gestational E2 increase

enhances liver repair,40 suggesting a physiological benefit for E2 during periods of liver

growth. Indeed, gper1-/- mutants exhibit significantly delayed liver outgrowth during

development and following injury. Nevertheless, gper1-/- mutants have a greater capacity

for liver regeneration compared to mtor-/- mutants, indicating that E2-GPER1 is one

among several inputs integrated by mTOR to regulate liver regeneration. We postulate

that GPER1 mediates E2 activation of PI3K-mTOR in a sex-dimorphic fashion to promote

liver regeneration.

Sexual dimorphism in liver cancer has long been documented, but a detailed mechanistic

understanding has not been established. Both protective and tumor-promoting effects for

E2 have been reported: E2-mediated reduction of inflammatory cytokines, such as IL-6 in

liver-resident immune cells, appears to inhibit tumorigenesis.41, 42 Several other studies

have shown liver tumor-promoting properties of E2.43, 44 Clinical trials in HCC patients

using the ESR antagonist tamoxifen have been disappointing;45, 46 in fact, tamoxifen

increased the size and number of hepatic lesions.47 Importantly, tamoxifen and

fulvestrant, used in our study, were subsequently found to be agonists for GPER17, 30, 48

explaining the negative clinical results, consistent with our data for fulvestrant inducing

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Akt phosphorylation in vivo, and increasing liver tumor burden. These findings are of

particular importance as men with cirrhosis have persistently elevated serum levels of E2,

and these patients are at highest risk for developing liver cancer. Our results definitively

demonstrate a growth-promoting role for E2 in liver cancer acting in hepatocytes through

GPER1; further studies are needed to define the various and likely receptor- and cell-type

specific influences of E2 on inflammation in the context of liver cancer.

The involvement of GPER1-PI3K-mTOR signaling in hepatocyte proliferation and organ

growth prompted us to hypothesize that GPER1 may promote liver carcinogenesis.

Indeed, activation of PI3K-Akt (~70%) and mTORC1 (~45%) pathways is found in HCC

and positively correlates with tumor metastasis, recurrence, and poor prognosis.49, 50

Genetic alterations of PI3K/mTOR pathway components are found at lower frequencies in

HCC: exome sequencing revealed mutation frequencies in PIK3CA (≤ 2%), mTOR (≤

2%), TSC1/TSC2 (≤ 5%), and PTEN (≤ 3%).51, 52 In the context of our data, is tempting to

speculate that PI3K-mTOR activity in HCC may instead depend upon upstream ligand

activation, mediated by increased E2 levels and GPER1 expression in cirrhotics, as also

observewd by Wei et al. 41 As >80% of HCC patients are diagnosed at late stages without

hope for cure, there is an urgent need for earlier detection and targeted therapeutic

interventions. The pro-proliferative consequences of E2-GPER1 activation of PI3K-mTOR

signaling, together with our in vivo data showing strong responses to GPER1 antagonist

treatment in both cancer initiation and progression, clearly indicate that drugs targeting

E2-GPER1 may offer exciting new therapeutic applications in liver cancer prevention and

treatment.

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Figure Legends

Figure 1. E2 increases liver size

(A) Brightfield and fluorescent images of male and female Tg(fabp10a:GFP) adult

zebrafish exposed to DMSO (0.1%) or E2 (10µM) daily for 6 weeks. Scale bars, 2mm. (B)

Liver weight:body weight (%). (C) Transcriptomic analysis showing E2-induced

upregulated genes (fold-change>10), particularly genes involved in cell cycle. (D) Polar

metabolomics demonstrates significant sex-dimorphic differences between DMSO and

E2-exposed livers (fold-change ≥2). (E) Liver size at 120hpf determined by fluorescence

microscopy in Tg(fabp10a:GFP) reporters, and by ISH for fabp10a. Scale bars, 200µm.

Quantification of fabp10a expression by qRT-PCR in Tg(fabp10a:GFP) larvae at 120hpf

(F), liver volume by confocal microscopy (G), and GFP+ hepatocyte number by FACS (H).

(I) Liver size after exposure to E2 (10µM), ANAS (10µM), and E2 + ANAS (10µM,15µM)

from 110-115hpf (10µM). Scale bar, 50µm. (J) Relative liver volume (fold-change from

DMSO) as assessed by confocal microscopy in Tg(fabp10a:GFP) larvae at 120hpf. ≥3

independent experiments, except (C) and (D) represent one experiment with ≥3 biological

replicates. Mean±standard error of the mean (SEM), n as indicated, ∗p<0.05, ∗∗p<0.01,

∗∗∗∗p<0.0001, two-tailed Student’s t-test.

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Figure 2. G protein-coupled estrogen receptor 1 (GPER1) mediates the estrogenic

effects on liver growth

(A) Liver size of larvae exposed to selective antagonists for ESR1 (MPP), ESR2 (PHTPP)

or GPER1 (G-15) alone and with E2 from 110-115hpf. (B) Liver area (fold-change from

DMSO). ∗∗∗∗p<0.0001, ns=not significant, two-tailed Student’s t-test. (C) Liver size of WT,

gper1 morphants ± human gper1 mRNA. (D) Relative liver size distribution. ∗p<0.05,

∗∗p<0.01, two-tailed Student’s t-test. (E) gper1-/-;Tg(fabp10a:GFP) fish reveal

progressively impaired liver development from 72-120 hpf. (F) Liver size of gper1+/+ and

gper1-/- larvae at 3, 4, 5 dpf, ns=not significant, ∗∗∗∗p<0.0001, two-way ANOVA. (G) gper1-

/- mutants failed to respond to E2 or G-1. (H) Liver area (fold-change from DMSO).

∗∗∗p=0.0005, ∗∗∗∗p<0.0001, ns=not significant, two-way ANOVA. Liver area assessed by

ISH for fabp10a at 120hpf. Values represent ≥3 independent experiments, mean±SEM, n

as indicated, scale bars, 200µm.

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Figure 3. E2 signals via GPER1 to promote cell cycle progression, proliferation, and cell

size increase in the liver

(A) FACS profiles of cell cycle analysis of GFP+ hepatocytes in Tg(fabp10a:GFP) larvae.

(B) Relative distribution of GFP+ hepatocytes in G1(light grey), S(black), or G2/M(dark

grey). ∗ difference in S- and G2/M-phase vs. DMSO-exposed hepatocytes, ∗∗p<0.01,

∗∗∗p<0.001, ns=not significant, two-tailed Student’s t-test. (C,D) Cell-cycle analysis

revealed decreased S-phase in gper1-/- mutants at 120hpf. ∗∗p<0.01, two-tailed Student’s

t-test. (E) PCNA (top panel) and TUNEL (bottom panel) staining of whole larvae section.

Liver outline in red. Insets show higher magnification of hepatocytes. Scale bars, 100µm,

scale bar (inset) 30µm. (F) % PCNA+ cells from total hepatocytes in the liver area.

∗∗∗p<0.001, ∗∗∗∗p<0.0001, two-tailed Student’s t-test; ∗∗∗∗p<0.0001, one-way ANOVA. (G)

Liver sections of DMSO, E2, or G-1-exposed larvae stained with DAPI (blue) and Pan-

Cadherin (red). Scale bars, 25µm. (H) Hepatocyte size quantification based on Pan-

Cadherin staining and cell area (fold-change from DMSO). ∗p<0.05, ∗∗p<0.01, two-tailed

Student’s t-test. Mean of 3 independent experiments±SEM, n as indicated.

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Figure 4. E2 signals via GPER1 to stimulate PI3K/mTORC1 to increase liver size

(A) Liver size in gper1+/+ and gper1-/- larvae. E2-induced liver size increase was blocked

by E2+MK-2206 co-exposure (top row arrowheads). Smaller liver in gper1-/- mutants was

normalized by 740Y-P (bottom row arrowheads). (B) Liver area (fold-change from

DMSO). † indicates difference from DMSO-exposed liver, ††††p<0.0001, ns=not significant,

two-way ANOVA. (C) Immunoblot analysis of gper1+/+ and gper1-/- whole larvae. (D) E2 or

G-1 exposure increased liver size in gper1+/+, mtor+/-, but not in gper1-/-, mtor-/-, or gper1-/-

;mtor-/- larvae (red arrowheads). (E) Liver area (fold from DMSO). ∗∗∗∗p<0.0001, ns=not

significant, two-way ANOVA. (F) Immunoblot analysis of gper1+/+, gper1-/-, mtor+/-, and

mtor-/- whole larvae. Results represent at ≥3 independent experiments. Representative

blots of ≥5 independent experiments. Liver area was assessed by ISH for fabp10a at

120hpf. Mean±SEM, n as indicated, all scale bars, 200µm.

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Figure 5. E2 promotes liver regeneration via activation of GPER1 and mTORC1

pathways

(A) Scheme for Mtz exposure and liver regeneration analysis. (B) gper1+/+, gper1-/-,

mtor+/-, and mtor-/- larvae in Tg(fabp10a:CFP-NTR) background at 150hpf. Scale bars,

200µm. (C) Liver area (fold-change from -Mtz WT) as determined by CFP expression. †

indicates significant difference from Mtz-treated WT at 30hpT, ††p<0.005, ††††p<0.0001,

two-way ANOVA.

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Figure 6. E2 signaling via GPER1 promotes sex dimorphism in adult liver growth

(A) Adult gper1+/+ and gper1-/- exposed to DMSO or E2. Scale bars, 2mm. (B) Liver

weight/body weight (%). ∗**∗p<0.0001, ns=not significant, two-way ANOVA. (C) Sex-

specific gper1 liver expression (vs. elf1α). ∗∗∗∗p<0.0001, two-tailed Student’s t-test. (D)

Immunoblot for p-Akt, GPER1, and β-actin levels in gper1+/+ and gper1-/- male livers. (E)

Immunofluorescent staining of DMSO or E2-exposed male donor-derived hepatocytes

with EdU (red) and HNF4α (green). White arrowheads indicate EdU and HNF4α double-

positive cells. Scale bar, 100µm. (F) % EdU+ hepatocytes. ∗ indicates significant

difference from DMSO controls. ∗p<0.05, ∗∗p<0.01, two-tailed Student’s t-test. (G)

Immunofluorescent staining of DMSO or E2-exposed primary hepatocytes with p-Akt

(pink) and CK18 (red). (H) Quantification of p-Akt intensity (AU) in DMSO or E2-treated

hepatocytes. ∗∗∗∗p<0.0001, two-tailed Student’s t-test. (I) Immunoblot for p-AKT, AKT, p-

S6, S6, and β-ACTIN levels in DMSO or E2-exposed male donor-derived hepatocytes.

Results represent ≥3 independent experiments, mean±SEM, n as indicated.

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Figure 7. E2 signaling promotes liver cancer initiation and progression via GPER1

(A) Human non-cirrhotic liver, cirrhotic liver, and HCC sections immunostained for

GPER1 in. Scale bar, 100µm. All values represent mean ± SEM, n as indicated. (B)

Quantification of GPER1 staining scores of non-cirrhotic livers, cirrhotic livers, adjacent

non-tumor (ANT) tissues, which are mostly cirrhotic, and HCC tissues. n as indicated,

mean ± SEM, �p<0.05, ns=not significant, two-tailed Mann-Whitney test. (C) Total liver

cancer incidence in DMBA-exposed adults at 7 months post fertilization (mpf) was

reduced by 50% in gper1-/- mutants compared to WT. p<0.05, one-tailed Chi-Square test.

(D) Number of liver tumors per fish was decreased in gper-/- mutants. ∗p<0.05, one-tailed

Mann-Whitney test. (E) Tumor size (mm) per fish. ∗∗p<0.01, two-tailed Student’s t-test. (F)

Scheme illustrating DMBA carcinogenesis followed by chemical prevention trial. (G)

Kaplan–Meier survival plot. n = 52, 66, 39 for DMSO, E2, and E2+G-15 treated adults at

249 days post first treatment, p<0.0001, Log-rank (Mantel-Cox) test. (H) Liver tumor size

(mm) per fish at 9 mpf. *p=0.0172, **p=0.0053, ****p<0.0001, ns=not significant, one-way

ANOVA, stratified by gender.

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Summary: E2 signals via GPER1 to increase embryonic and adult liver size and growth by activation of PI3K signaling to mTOR. E2 signaling via GPER1 contributes to sex-dimorphic adult hepatocyte proliferation and promotes liver cancer formation and progression What you need to know Background: We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Findings: Exposure to 17 beta-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. GPER1 is a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. HCC samples from patients had increased levels of GPER1, compared with non-tumor tissues Limitations: This study was performed in an animal model of HCC. Implications for patients: Inhibitors of GPER1 might be developed for liver cancer prevention or treatment.

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Supporting Documents

Supplementary Figure Legends

Supplementary Figure S1. Estrogen increases liver growth and size.

(A) Venn diagram showing intersection of genes differentially expressed in the livers of male

vs. male+E2, female vs. female+E2, and female vs. male. (fold change >10). Values

demonstrate number of genes upregulated by E2 in male liver (299 genes), female liver (125

genes) or female-associated liver genes (75 genes). (B) Heatmap representing genes

significantly enriched upon E2 treatment compared to sex-matched DMSO control of male

and female livers (fold change > 10 from DMSO). n=2 for male, female, male+E2, and

female+E2. Scaled expression value is plotted in green-red color scale with green indicating

low expression and red representing high expression. Hierarchical clustering analysis is

based on Pearson correlation. (C) Transcriptomic analysis showing E2-induced upregulated

genes in female only livers (fold change >10 from DMSO). Gene ontology analysis revealed

upregulation of genes involved in convergent extension. (D) Heatmap representing polar

metabolite abundance as determined by LC–MS/MS of male and female livers with and

without E2, n=5 for male, female and female+E2, and male+E2. Scaled metabolite

abundance value is plotted in green-red color scale with green indicating low abundance and

red representing high abundance. Hierarchical clustering is based on Pearson correlation. (E)

Polar metabolomics analysis demonstrating significant sex dimorphic differences between

DMSO and E2-exposed female only livers (fold change ≥2 from DMSO). Metabolic pathway

analysis indicated regulation of ammonia and glutamate metabolism.

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Supplementary Figure S2. G protein-coupled estrogen receptor 1(GPER1) mediates the

estrogenic effects on liver growth.

(A) Representative images of WT larvae with increased liver size upon E2 or G-1 treatments,

decreased liver size upon G-15 treatment, and normalized liver size upon E2 and G-15 co-

treatment as assayed by ISH for fabp10a at 120 hpf. Scale bar 200 µm. (B) Quantification of

GFP+ hepatocytes in fabp10a:GFP larvae by FACS (fold from DMSO). n=3 independent

experiments of 30 pooled larvae, mean ± SEM, ���p<0.001, ����p<0.0001, two-tailed

Student’s t-test; ����p<0.0001, one-way ANOVA. (C) Quantification of liver area as

determined by ISH for fabp10a (fold from DMSO). n as indicated, mean ± SEM, ��p<0.01,

����p<0.0001, ns=not significant, two-tailed Student’s t-test. (D) Quantification of liver area in

zebrafish larvae injected with GPER1 mRNA, treated with ANAS (10µM), or GPER1 mRNA +

ANAS (10µM) as determined by ISH for fabp10a (fold from DMSO). n as indicated, mean ±

SEM, ��p=0.06, ����p<0.0001, ns=not significant, two-way ANOVA. (E) Quantification of

liver area in zebrafish larvae treated with DMSO, E2 (10µM) or BPA (25, 50µM) as

determined by ISH for fabp10a (fold from DMSO). n as indicated, mean ± SEM, �p<0.05,

��p<0.01, two-tailed Student’s t-test. (F) Quantification of liver area in zebrafish larvae treated

with DMSO, E2 (10µM) or TAM (10, 20, 30µM) as determined by ISH for fabp10a (fold from

DMSO). n as indicated, mean ± SEM, �p<0.05, ��p<0.01, ���p<0.001, two-tailed Student’s t-

test. (G) Representative images of Tg(5xERE:GFP;fabp10a:mKate) reporter fish exposed to

DMSO, 5hrs of E2, 24hrs of E2 at 120 hpf. Scale bar 80 µm. (H) Quantification GFP intensity

(AU) of Tg(5xERE:GFP;fabp10a:mKate) reporter fish. n as indicated, mean ± SEM,

��p<0.01, ����p<0.0001, two-tailed Student’s t-test.

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Supplementary Figure S3. GPER1 mediates the estrogenic effects on liver growth.

(A) Lateral and dorsal view of gper1 expression in larvae at 72, 96, 120 hpf after ISH for

gper1. gper1 is expressed in the liver (red arrowhead) starting at 96 hpf. Scale bars 200 µm.

(B) Expression of gper1 transcripts in whole embryo at 24, 48, 72, 96, 109 and 120 hpf as

measured by RT-PCR compared to elf1α housekeeping gene control. (C) Expression of

gper1 transcripts in whole embryo at 12, 35, 48, 72, and 120 hpf as measured by quantitative

RT-PCR (fold from gper1 expression at 12 hpf). (D) Genomic organization of zebrafish gper1.

Black boxes represent exons with ATG site indicated by the arrow. Sequence alignment of

gper1+/+ siblings and gper1-/- mutant showing TALEN generated 29 base pair deletion leading

to premature stop codon (*). (E) Immunoblot analysis of GPER1 (red arrow) and β-actin

levels showing loss of GPER1 in gper1-/- mutants compared to gper1+/+ siblings. (F)

Representative images of gper1-/- and gper1+/+ larvae ISH for foxA3 (endoderm) and prox1

(hepatic progenitor) at 48 hpf, prox1 and fabp10a (hepatocyte) at 72 hpf, fabp10a and deltaC

(biliary tree) at 120 hpf. Decreased expression of fabp10a and deltaC in gper1-/- compared to

gper1+/+ was observed only at 120 hpf. Scale bars 200 µm. (G) Quantification of marker

expression area in gper1-/- and gper1+/+ larvae as assessed by ISH (fold from gper1+/+). n as

indicated, mean ± SEM, ��p<0.01, ����p<0.0001, ns = not significant, two-tailed Student’s t-

test.

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Supplementary Figure S4. Estrogen increases cell proliferation in the liver.

(A,B) BrdU analysis of whole larvae exposed to DMSO or E2 at 120 hpf. Larvae were pulsed

with BrdU and stained for BrdU after fixation. Larvae treated with E2 had higher number of

BrdU positive cells per fixed liver area compared to DMSO-exposed controls. Scale bar 400

µm, scale bar (inset) 100 µm, n as indicated, mean ± SEM, ���p<0.001, two-tailed Mann-

Whitney test. (C) Cell cycle analysis of whole larvae 45hr after treated with DMSO or E2 for

5hr in G1(light grey), S(black), or G2/M(dark grey)-phase. ns=not significant, two-tailed

Student’s t-test. (D) PCNA staining of liver larvae sections. Scale bar 15µm. (E) % PCNA+

cells from total hepatocytes in the liver area in larvae 0hr, 15hr and 45hr after treated with

DMSO or E2 for 5hr. n as indicated, mean ± SEM, ∗p<0.05, ∗∗∗∗p<0.0001, ns=not significant,

two-tailed Student’s t-test. (F) TUNEL staining of liver larvae sections. Scale bar 15µm.

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Supplementary Figure S5. Estrogen signals through GPER1 to activate PI3K/mTOR

pathway to increase liver size.

(A) Liver size of WT larvae after chemical exposures as assessed by ISH for fabp10a at 120

hpf. E2 exposure increased liver size (27%; 21/78, 21 larvae with phenotype out of 78 total

larvae observed) while PI3K inhibitor LY292002 decreased liver size (72%; 55/76). Co-

treatment of E2 and LY292002 blocked estrogenic effect on liver size (86%; 59/69). (B) Liver

size of WT and gper1MO knockdown larvae after chemical exposures at 120 hpf as assayed

by ISH for fabp10a. gper1 morphants had decreased liver size (60%; 15/26). Treatment of

larvae with the PI3K-activator 740Y-P increased liver size (77%; 24/31) and rescued small

liver phenotype in gper1 morphants (61%; 11/18). (C) PCNA staining of liver larvae section.

Scale bars, 15µm. (D) % PCNA+ cells from total hepatocytes in the liver area. ∗p<0.05,

∗∗∗∗p<0.0001, two-tailed Student’s t-test; (E) Liver size of gper1+/+ and gper1-/- larvae ISH for

fabp10a at 120 hpf after chemical exposures. E2 or G-1-exposed gper1+/+ larvae had

increased liver size (red arrowhead) that can be blocked by co-exposure of E2+Rapamycin or

G-1+Rapamycin. gper1-/- blocked E2 and G-1 effects on liver size. (F) Liver area of gper1+/+

and gper1-/- larvae ISH for fabp10a at 120 hpf after chemical exposures (fold from DMSO). n

as indicated, mean ± SEM, **p=0.0323 vs. WT cont, ∗∗∗∗p<0.0001, ns = not significant, two-

way ANOVA. (G) Liver size distribution of WT larvae and mtor morphants as assessed by

ISH for fabp10a at 120 hpf upon exposure to DMSO or E2 as % of larvae with large (dark

green), medium (light green) or small (grey) livers. (H) Liver size of WT and mtor morphants

as determined by ISH for lfabp10a at 120 hpf. (I) Whole mount WT larvae at 120 hpf

immunostained for p-Akt and p-S6 upon DMSO or E2 exposure. Liver is outlined in black. All

scale bars 200 µm.

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Supplementary Figure S6. GPER1 is essential regeneration and for male-biased response

of estrogen on liver size.

(A) Whole-mount p-S6 immunostaining revealed mTORC1 activation in regenerating WT

livers but not in gper1-/- or mtor-/- livers. Scale bar, 200 µm, insets 20 µm. (B) Quantification of

p-S6 intensity (AU) from insets in gper1+/+, gper1-/-, mtor+/-, and mtor-/- larval livers ±Mtz.

∗p=0.00568, **p<0.0001, two-way ANOVA. 3 independent experiments, mean±SEM, n as

indicated.

(C) Liver weight (mg) of gper1+/+ and gper1-/-, male and female adult fish treated with DMSO

or E2 (10µM) for 6 weeks. (D) Body weight (mg) of gper1+/+ and gper1-/-, male and female

adult fish treated with DMSO or E2 (10µM) for 6 weeks. (E) Body length (cm) of gper1+/+ and

gper1-/-, male and female adult fish treated with DMSO or E2 (10µM) for 6 weeks. (F) Liver

weight/body weight (%) of gper1+/+ and gper1-/-, male and female adult fish treated with

DMSO or ANAS (50µM) for 6 weeks. (C)-(F) *p<0.05, **p<0.01, ****p<0.0001, ns=not

significant, two-way ANOVA, gender stratified. (G) No significant differences were observed

from liver histology of gper1+/+ and gper1-/-, male and female adult fish treated with DMSO or

ANAS (50µM) for 6 weeks. (H) RNA sequencing data showing relative expression in

Fragments Per Kilobase of transcription per Million mapped reads (FPKM) of ESR1, ESR2,

and GPER1 in human primary hepatocytes and in mouse fibroblast co-cultured cells.

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Supplementary Figure S7. Activation of E2/GPER1 signaling promotes male liver cancer

initiation and progression.

(A) Signaling responses in HepG2 cells to E2 and/or G-15 exposure. (B) Representative

images of GPER1 staining scoring system. 0 = minimal or no staining; 1+ = faint/mild

staining; 2+ = moderate/strong staining. Scale bar 50 µm. (C) Quantification of GPER1

staining scores in non-cirrhotic male and female livers. n as indicated, mean ± SEM. (D)

Quantification of liver tumor size (mm) per fish in DMBA-exposed WT fish followed by DMSO,

E2, E2+G-15, or E2+Ful (Fulvestrant) exposure up until 9 months post fertilization. n as

indicated, mean ± SEM, �p < 0.05, ��P < 0.01, ns=not significant, two-tailed Student’s t-test.

(E) Akt signaling responses to E2 and fulvestrant (Ful; 5µM) exposure from 110-115 hpf in

pooled zebrafish larvae at 120 hpf. (F) Tg(fabp10a:GFP) fish after chemical treatments and

corresponding liver histology. NT=non-tumor, scale bars (whole animal) 2mm, scale bar

(histology section) 100µm. (G) Histological features of adult zebrafish liver stained with

hematoxylin and eosin (H&E). Top panel shows zoomed images from normal male liver (a),

normal female liver (b), and livers with hepatocellular adenoma (HCA) (c), hepatocellular

carcinoma (HCC) (d), and cholangiocarcinoma (CC) (e). Scale bar, 25 µm. Bottom panel

demonstrates low magnification images of normal male liver (f), normal female liver (g), and

livers with large tumor (h), small tumor (i) and multi-foci tumor (j). Scale bars (f)-(i) 100 µm,

scale bar (j) 250 µm.

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Supplementary Methods

Primary human hepatocyte culture and cell culture experiments

96-well glass bottom plates (Greiner Bio-one) were incubated with type I collagen solubilized

in water (100 µg/ml; BD Biosciences) for 1h at 37 °C. A J2-3T3 fib roblast feeder layer (gift

from H. Green) was plated onto collagen at a density of 16,000 cells/well at day -1. Primary

human hepatocytes (Hu4175:3-yr old male donor, Invitrogen) were plated onto fibroblasts on

day 0 at a density of 4,000 cells/well and maintained under standard culture conditions with

daily replacement of hepatocyte medium (1X DMEM/F12 (phenol red free), 10% fetal bovine

serum (FBS), 15.6 ug/ml insulin, 7.5 µg/ml, hydrocortisone, 16 ng/ml glucagon, 1% penicillin-

streptomycin). From day 6, FBS concentration of the hepatocyte medium was lowered to 2%

and DMSO concentration was set at 0.1%. From day 7-10, cells were pulsed daily with E2 for

5.5h/day, followed by 18.5h culture in hepatocyte medium with 10µM EdU. On day 11, cells

were fixed in 4% PFA and stained with Click-iT EdU Alexa Fluor 594 Imaging Kit (Thermo

Fisher), with additional antibody staining for HNF4α (ab55223) and goat-anti-mouse 488

(Jackson Immunolabs). Nuclei were visualized with Hoechst stain (Invitrogen). Cells were

imaged and analyzed in NIS-Elements Viewer 4.0, ImageJ, and Photoshop.

Cell culture

HepG2 cells (Sigma-Aldrich, USA) were cultured in phenol-red free, Minimum Essential

Medium Eagle (MEM) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone,

Thermo Scientific, USA), L-glutamine (2mM), 1% non-essential amino acids and penicillin-

streptomycin (10,000 U/mL), at 37°C and 5% CO2. Fol lowing 24 h of serum starvation,

HepG2 cells were incubated with DMSO or G15 (500 nM) for 20 min prior to exposure to E2

(10nM), or E2+G15 for 30 min

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Supplementary Statistical Analysis

2-Way ANOVA analysis was performed where appropriate. Resulting p values for interaction

effects for each figure are listed below:

Figure 2F:

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.017 <0.0001 **** Yes GPER1 Factor 88.56 <0.0001 **** Yes Age Factor 2.437 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1.202 2 0.6008 F (2, 414) = 46.48 P<0.0001 GPER1 Factor 104.6 2 52.31 F (2, 414) = 4046 P<0.0001 Age Factor 2.878 1 2.878 F (1, 414) = 222.7 P<0.0001 Residual 5.352 414 0.01293

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

3dpf:gper1 +/+ vs. 3dpf:gper1 -/- 0.02061 -0.03554 to 0.07677 No ns 0.9002

3dpf:gper1 +/+ vs. 4dpf:gper1 +/+ -0.6682 -0.7344 to -0.6019 Yes **** <0.0001

3dpf:gper1 +/+ vs. 4dpf:gper1 -/- -0.4395 -0.4947 to -0.3842 Yes **** <0.0001

3dpf:gper1 +/+ vs. 5dpf:gper1 +/+ -1.446 -1.514 to -1.378 Yes **** <0.0001

3dpf:gper1 +/+ vs. 5dpf:gper1 -/- -1.164 -1.222 to -1.107 Yes **** <0.0001

3dpf:gper1 -/- vs. 4dpf:gper1 +/+ -0.6888 -0.7477 to -0.6299 Yes **** <0.0001

3dpf:gper1 -/- vs. 4dpf:gper1 -/- -0.4601 -0.5063 to -0.4139 Yes **** <0.0001

3dpf:gper1 -/- vs. 5dpf:gper1 +/+ -1.467 -1.527 to -1.406 Yes **** <0.0001

3dpf:gper1 -/- vs. 5dpf:gper1 -/- -1.185 -1.234 to -1.136 Yes **** <0.0001

4dpf:gper1 +/+ vs. 4dpf:gper1 -/- 0.2287 0.1706 to 0.2868 Yes **** <0.0001

4dpf:gper1 +/+ vs. 5dpf:gper1 +/+ -0.7779 -0.8481 to -0.7076 Yes **** <0.0001

4dpf:gper1 +/+ vs. 5dpf:gper1 -/- -0.496 -0.5562 to -0.4357 Yes **** <0.0001

4dpf:gper1 -/- vs. 5dpf:gper1 +/+ -1.007 -1.067 to -0.9465 Yes **** <0.0001

4dpf:gper1 -/- vs. 5dpf:gper1 -/- -0.7247 -0.7726 to -0.6768 Yes **** <0.0001

5dpf:gper1 +/+ vs. 5dpf:gper1 -/- 0.2819 0.2198 to 0.344 Yes **** <0.0001

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Figure 2H:

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 4.535 0.0002 *** Yes GPER1 Factor 13.09 <0.0001 **** Yes Treatments Factor 32.08 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.52 2 0.26 F (2, 182) = 8.847 P=0.0002 GPER1 Factor 1.502 2 0.7508 F (2, 182) = 25.55 P<0.0001 Treatments Factor 3.679 1 3.679 F (1, 182) = 125.2 P<0.0001 Residual 5.349 182 0.02939

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.1611 0.05204 to 0.2702 Yes *** 0.0005

DMSO:gper1 +/+ vs. E2:gper1 +/+ -0.3569 -0.4816 to -0.2322 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1 -/- 0.07512 -0.08651 to 0.2367 No ns 0.7631

DMSO:gper1 +/+ vs. G-1:gper1 +/+ -0.2417 -0.3635 to -0.1199 Yes **** <0.0001

DMSO:gper1 +/+ vs. G-1:gper1 -/- 0.09102 -0.01674 to 0.1988 No ns 0.1505

DMSO:gper1 -/- vs. E2:gper1 +/+ -0.518 -0.6439 to -0.3921 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 -/- -0.08602 -0.2485 to 0.0765 No ns 0.649

DMSO:gper1 -/- vs. G-1:gper1 +/+ -0.4028 -0.5258 to -0.2799 Yes **** <0.0001

DMSO:gper1 -/- vs. G-1:gper1 -/- -0.07012 -0.1792 to 0.03897 No ns 0.4357

E2:gper1 +/+ vs. E2:gper1 -/- 0.432 0.2586 to 0.6054 Yes **** <0.0001

E2:gper1 +/+ vs. G-1:gper1 +/+ 0.1152 -0.02188 to 0.2522 No ns 0.1547

E2:gper1 +/+ vs. G-1:gper1 -/- 0.4479 0.3232 to 0.5726 Yes **** <0.0001

E2:gper1 -/- vs. G-1:gper1 +/+ -0.3168 -0.4881 to -0.1455 Yes **** <0.0001

E2:gper1 -/- vs. G-1:gper1 -/- 0.0159 -0.1457 to 0.1775 No ns 0.9998

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Figure 4B:

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 8.979 <0.0001 **** Yes GPER1 Factor 21.14 <0.0001 **** Yes Treatments Factor 28.76 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 2.284 5 0.4567 F (5, 201) = 10.44 P<0.0001 GPER1 Factor 5.377 5 1.075 F (5, 201) = 24.58 P<0.0001 Treatments Factor 7.315 1 7.315 F (1, 201) = 167.2 P<0.0001 Residual 8.793 201 0.04375

.

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.1484 -0.04979 to 0.3466 No ns 0.361

DMSO:gper1 +/+ vs. G-1:gper1 +/+ -0.5409 -0.7451 to -0.3368 Yes **** <0.0001

DMSO:gper1 +/+ vs. G-1:gper1 -/- 0.07811 -0.1107 to 0.2669 No ns 0.9682

DMSO:gper1 +/+ vs. E2:gper1 +/+ -0.4441 -0.6815 to -0.2067 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1 -/- 0.2086 -0.04509 to 0.4622 No ns 0.2247

DMSO:gper1 +/+ vs. MK:gper1 +/+ 0.1573 -0.06273 to 0.3772 No ns 0.4355

DMSO:gper1 +/+ vs. MK:gper1 -/- 0.2649 0.03977 to 0.49 Yes ** 0.0074

DMSO:gper1 +/+ vs. MK+E2:gper1 +/+ -0.1945 -0.4196 to 0.03061 No ns 0.1648

DMSO:gper1 +/+ vs. MK+E2:gper1 -/- 0.2754 0.08223 to 0.4685 Yes *** 0.0003

DMSO:gper1 +/+ vs. 740YP:gper1 +/+ -0.4384 -0.6315 to -0.2452 Yes **** <0.0001

DMSO:gper1 +/+ vs. 740YP:gper1 -/- -0.04803 -0.3242 to 0.2282 No ns >0.9999

DMSO:gper1 -/- vs. G-1:gper1 +/+ -0.6893 -0.9083 to -0.4703 Yes **** <0.0001

DMSO:gper1 -/- vs. G-1:gper1 -/- -0.07029 -0.275 to 0.1344 No ns 0.9927

DMSO:gper1 -/- vs. E2:gper1 +/+ -0.5925 -0.8428 to -0.3422 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 -/- 0.06016 -0.2056 to 0.3259 No ns 0.9998

DMSO:gper1 -/- vs. MK:gper1 +/+ 0.008849 -0.225 to 0.2427 No ns >0.9999

DMSO:gper1 -/- vs. MK:gper1 -/- 0.1165 -0.1222 to 0.3551 No ns 0.9022

DMSO:gper1 -/- vs. MK+E2:gper1 +/+ -0.3429 -0.5815 to -0.1042 Yes *** 0.0002

DMSO:gper1 -/- vs. MK+E2:gper1 -/- 0.127 -0.08181 to 0.3357 No ns 0.6854

DMSO:gper1 -/- vs. 740YP:gper1 +/+ -0.5868 -0.7955 to -0.378 Yes **** <0.0001

DMSO:gper1 -/- vs. 740YP:gper1 -/- -0.1964 -0.4838 to 0.09095 No ns 0.5082

G-1:gper1 +/+ vs. G-1:gper1 -/- 0.6191 0.4085 to 0.8296 Yes **** <0.0001

G-1:gper1 +/+ vs. E2:gper1 +/+ 0.09681 -0.1582 to 0.3519 No ns 0.9835

G-1:gper1 +/+ vs. E2:gper1 -/- 0.7495 0.4793 to 1.02 Yes **** <0.0001

G-1:gper1 +/+ vs. MK:gper1 +/+ 0.6982 0.4593 to 0.9371 Yes **** <0.0001

G-1:gper1 +/+ vs. MK:gper1 -/- 0.8058 0.5622 to 1.049 Yes **** <0.0001

G-1:gper1 +/+ vs. MK+E2:gper1 +/+ 0.3464 0.1028 to 0.5901 Yes *** 0.0003

G-1:gper1 +/+ vs. MK+E2:gper1 -/- 0.8163 0.6019 to 1.031 Yes **** <0.0001

G-1:gper1 +/+ vs. 740YP:gper1 +/+ 0.1026 -0.1118 to 0.317 No ns 0.9137

G-1:gper1 +/+ vs. 740YP:gper1 -/- 0.4929 0.2014 to 0.7844 Yes **** <0.0001

G-1:gper1 -/- vs. E2:gper1 +/+ -0.5222 -0.7651 to -0.2793 Yes **** <0.0001

G-1:gper1 -/- vs. E2:gper1 -/- 0.1305 -0.1283 to 0.3893 No ns 0.8809

G-1:gper1 -/- vs. MK:gper1 +/+ 0.07914 -0.1468 to 0.305 No ns 0.9914

G-1:gper1 -/- vs. MK:gper1 -/- 0.1868 -0.04413 to 0.4177 No ns 0.2466

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G-1:gper1 -/- vs. MK+E2:gper1 +/+ -0.2726 -0.5035 to -0.04171 Yes ** 0.007

G-1:gper1 -/- vs. MK+E2:gper1 -/- 0.1973 -0.002591 to 0.3971 No ns 0.0567

G-1:gper1 -/- vs. 740YP:gper1 +/+ -0.5165 -0.7163 to -0.3166 Yes **** <0.0001

G-1:gper1 -/- vs. 740YP:gper1 -/- -0.1261 -0.4071 to 0.1548 No ns 0.9432

E2:gper1 +/+ vs. E2:gper1 -/- 0.6527 0.3565 to 0.9489 Yes **** <0.0001

E2:gper1 +/+ vs. MK:gper1 +/+ 0.6014 0.3335 to 0.8693 Yes **** <0.0001

E2:gper1 +/+ vs. MK:gper1 -/- 0.709 0.4369 to 0.9811 Yes **** <0.0001

E2:gper1 +/+ vs. MK+E2:gper1 +/+ 0.2496 -0.02247 to 0.5217 No ns 0.1066

E2:gper1 +/+ vs. MK+E2:gper1 -/- 0.7195 0.4732 to 0.9658 Yes **** <0.0001

E2:gper1 +/+ vs. 740YP:gper1 +/+ 0.005777 -0.2405 to 0.2521 No ns >0.9999

E2:gper1 +/+ vs. 740YP:gper1 -/- 0.3961 0.0804 to 0.7118 Yes ** 0.0028

E2:gper1 -/- vs. MK:gper1 +/+ -0.05131 -0.3337 to 0.2311 No ns >0.9999

E2:gper1 -/- vs. MK:gper1 -/- 0.05631 -0.2301 to 0.3427 No ns >0.9999

E2:gper1 -/- vs. MK+E2:gper1 +/+ -0.4031 -0.6894 to -0.1167 Yes *** 0.0004

E2:gper1 -/- vs. MK+E2:gper1 -/- 0.0668 -0.1952 to 0.3288 No ns 0.9995

E2:gper1 -/- vs. 740YP:gper1 +/+ -0.6469 -0.9089 to -0.3849 Yes **** <0.0001

E2:gper1 -/- vs. 740YP:gper1 -/- -0.2566 -0.5847 to 0.0715 No ns 0.2946

MK:gper1 +/+ vs. MK:gper1 -/- 0.1076 -0.1494 to 0.3647 No ns 0.9653

MK:gper1 +/+ vs. MK+E2:gper1 +/+ -0.3517 -0.6088 to -0.09471 Yes *** 0.0006

MK:gper1 +/+ vs. MK+E2:gper1 -/- 0.1181 -0.1114 to 0.3477 No ns 0.8656

MK:gper1 +/+ vs. 740YP:gper1 +/+ -0.5956 -0.8252 to -0.366 Yes **** <0.0001

MK:gper1 +/+ vs. 740YP:gper1 -/- -0.2053 -0.5081 to 0.09754 No ns 0.5216

MK:gper1 -/- vs. MK+E2:gper1 +/+ -0.4594 -0.7208 to -0.1979 Yes **** <0.0001

MK:gper1 -/- vs. MK+E2:gper1 -/- 0.01049 -0.224 to 0.245 No ns >0.9999

MK:gper1 -/- vs. 740YP:gper1 +/+ -0.7032 -0.9377 to -0.4688 Yes **** <0.0001

MK:gper1 -/- vs. 740YP:gper1 -/- -0.3129 -0.6195 to -0.006343 Yes * 0.0408

MK+E2:gper1 +/+ vs. MK+E2:gper1 -/- 0.4699 0.2354 to 0.7043 Yes **** <0.0001

MK+E2:gper1 +/+ vs. 740YP:gper1 +/+ -0.2439 -0.4783 to -0.009395 Yes * 0.0335

MK+E2:gper1 +/+ vs. 740YP:gper1 -/- 0.1465 -0.1601 to 0.453 No ns 0.9144

MK+E2:gper1 -/- vs. 740YP:gper1 +/+ -0.7137 -0.9177 to -0.5098 Yes **** <0.0001

MK+E2:gper1 -/- vs. 740YP:gper1 -/- -0.3234 -0.6073 to -0.03948 Yes * 0.0114

740YP:gper1 +/+ vs. 740YP:gper1 -/- 0.3903 0.1064 to 0.6742 Yes *** 0.0006

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Figure 4E:

Two-way ANOVA Ordinary Alpha 0.05

Source of Variation % of total variation P value

P value summary Significant?

Interaction 3.556 <0.0001 **** Yes GPER1 Factor 1.912 <0.0001 **** Yes Treatments Factor 76.97 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1.344 8 0.168 F (8, 196) = 5.922 P<0.0001 GPER1 Factor 0.7229 2 0.3615 F (2, 196) = 12.74 P<0.0001 Treatments Factor 29.1 4 7.275 F (4, 196) = 256.3 P<0.0001 Residual 5.562 196 0.02838

Tukey's multiple comparisons test

Mean Diff. 95.00% CI of diff. Significant?

Summary Adjusted P Value

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DMSO:+/+ vs. DMSO:-/- 0.1878 -0.04285 to 0.4184 No ns 0.2574 DMSO:+/+ vs. DMSO:+/+;+/- -0.05403 -0.2936 to 0.1856 No ns >0.9999 DMSO:+/+ vs. DMSO:+/+;-,- 0.6181 0.3652 to 0.8709 Yes **** <0.0001 DMSO:+/+ vs. DMSO:-/-;-/- 0.6099 0.3766 to 0.8432 Yes **** <0.0001 DMSO:+/+ vs. E2:+/+ -0.4318 -0.6847 to -0.179 Yes **** <0.0001 DMSO:+/+ vs. E2:-/- 0.1782 -0.07462 to 0.4311 No ns 0.5022 DMSO:+/+ vs. E2:+/+;+/- -0.3566 -0.5872 to -0.1259 Yes **** <0.0001 DMSO:+/+ vs. E2:+/+;-,- 0.6417 0.4055 to 0.878 Yes **** <0.0001 DMSO:+/+ vs. E2:-/-;-/- 0.6003 0.364 to 0.8365 Yes **** <0.0001 DMSO:+/+ vs. G-1:+/+ -0.4048 -0.6576 to -0.1519 Yes **** <0.0001 DMSO:+/+ vs. G-1:-/- 0.2116 -0.01663 to 0.4399 No ns 0.1018 DMSO:+/+ vs. G-1:+/+;+/- -0.1779 -0.3987 to 0.04276 No ns 0.2724 DMSO:+/+ vs. G-1:+/+;-,- 0.6256 0.3822 to 0.869 Yes **** <0.0001 DMSO:+/+ vs. G-1:-/-;-/- 0.6324 0.3736 to 0.8912 Yes **** <0.0001 DMSO:-/- vs. DMSO:+/+;+/- -0.2418 -0.4507 to -0.03295 Yes ** 0.0083 DMSO:-/- vs. DMSO:+/+;-,- 0.4303 0.2064 to 0.6542 Yes **** <0.0001 DMSO:-/- vs. DMSO:-/-;-/- 0.4221 0.2205 to 0.6237 Yes **** <0.0001 DMSO:-/- vs. E2:+/+ -0.6196 -0.8435 to -0.3956 Yes **** <0.0001 DMSO:-/- vs. E2:-/- -0.009545 -0.2335 to 0.2144 No ns >0.9999 DMSO:-/- vs. E2:+/+;+/- -0.5443 -0.7428 to -0.3458 Yes **** <0.0001 DMSO:-/- vs. E2:+/+;-,- 0.454 0.249 to 0.659 Yes **** <0.0001 DMSO:-/- vs. E2:-/-;-/- 0.4125 0.2075 to 0.6175 Yes **** <0.0001 DMSO:-/- vs. G-1:+/+ -0.5926 -0.8165 to -0.3686 Yes **** <0.0001 DMSO:-/- vs. G-1:-/- 0.02384 -0.1719 to 0.2196 No ns >0.9999 DMSO:-/- vs. G-1:+/+;+/- -0.3657 -0.5526 to -0.1788 Yes **** <0.0001 DMSO:-/- vs. G-1:+/+;-,- 0.4378 0.2246 to 0.651 Yes **** <0.0001 DMSO:-/- vs. G-1:-/-;-/- 0.4447 0.214 to 0.6753 Yes **** <0.0001 DMSO:+/+;+/- vs. DMSO:+/+;-,- 0.6721 0.4389 to 0.9053 Yes **** <0.0001 DMSO:+/+;+/- vs. DMSO:-/-;-/- 0.6639 0.4521 to 0.8757 Yes **** <0.0001 DMSO:+/+;+/- vs. E2:+/+ -0.3778 -0.6109 to -0.1446 Yes **** <0.0001 DMSO:+/+;+/- vs. E2:-/- 0.2323 -0.0009056 to 0.4654 No ns 0.0520 DMSO:+/+;+/- vs. E2:+/+;+/- -0.3025 -0.5114 to -0.09368 Yes *** 0.0001 DMSO:+/+;+/- vs. E2:+/+;-,- 0.6958 0.4807 to 0.9108 Yes **** <0.0001 DMSO:+/+;+/- vs. E2:-/-;-/- 0.6543 0.4392 to 0.8693 Yes **** <0.0001 DMSO:+/+;+/- vs. G-1:+/+ -0.3508 -0.5839 to -0.1176 Yes **** <0.0001 DMSO:+/+;+/- vs. G-1:-/- 0.2656 0.05942 to 0.4719 Yes ** 0.0015 DMSO:+/+;+/- vs. G-1:+/+;+/- -0.1239 -0.3218 to 0.07393 No ns 0.6984 DMSO:+/+;+/- vs. G-1:+/+;-,- 0.6796 0.4567 to 0.9025 Yes **** <0.0001 DMSO:+/+;+/- vs. G-1:-/-;-/- 0.6865 0.4469 to 0.9261 Yes **** <0.0001 DMSO:+/+;-,- vs. DMSO:-/-;-/- -0.008193 -0.2349 to 0.2185 No ns >0.9999 DMSO:+/+;-,- vs. E2:+/+ -1.05 -1.297 to -0.8031 Yes **** <0.0001 DMSO:+/+;-,- vs. E2:-/- -0.4398 -0.6866 to -0.1931 Yes **** <0.0001 DMSO:+/+;-,- vs. E2:+/+;+/- -0.9746 -1.199 to -0.7507 Yes **** <0.0001 DMSO:+/+;-,- vs. E2:+/+;-,- 0.02365 -0.2061 to 0.2534 No ns >0.9999 DMSO:+/+;-,- vs. E2:-/-;-/- -0.01781 -0.2475 to 0.2119 No ns >0.9999 DMSO:+/+;-,- vs. G-1:+/+ -1.023 -1.27 to -0.7761 Yes **** <0.0001 DMSO:+/+;-,- vs. G-1:-/- -0.4065 -0.6279 to -0.185 Yes **** <0.0001 DMSO:+/+;-,- vs. G-1:+/+;+/- -0.796 -1.01 to -0.5823 Yes **** <0.0001 DMSO:+/+;-,- vs. G-1:+/+;-,- 0.007514 -0.2296 to 0.2446 No ns >0.9999 DMSO:+/+;-,- vs. G-1:-/-;-/- 0.01437 -0.2385 to 0.2672 No ns >0.9999 DMSO:-/-;-/- vs. E2:+/+ -1.042 -1.268 to -0.815 Yes **** <0.0001 DMSO:-/-;-/- vs. E2:-/- -0.4317 -0.6583 to -0.205 Yes **** <0.0001 DMSO:-/-;-/- vs. E2:+/+;+/- -0.9664 -1.168 to -0.7649 Yes **** <0.0001 DMSO:-/-;-/- vs. E2:+/+;-,- 0.03185 -0.1761 to 0.2398 No ns >0.9999 DMSO:-/-;-/- vs. E2:-/-;-/- -0.009622 -0.2176 to 0.1984 No ns >0.9999 DMSO:-/-;-/- vs. G-1:+/+ -1.015 -1.241 to -0.788 Yes **** <0.0001

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Figure 5C:

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant?

DMSO:-/-;-/- vs. G-1:-/- -0.3983 -0.5971 to -0.1994 Yes **** <0.0001 DMSO:-/-;-/- vs. G-1:+/+;+/- -0.7878 -0.978 to -0.5977 Yes **** <0.0001 DMSO:-/-;-/- vs. G-1:+/+;-,- 0.01571 -0.2004 to 0.2318 No ns >0.9999 DMSO:-/-;-/- vs. G-1:-/-;-/- 0.02256 -0.2107 to 0.2558 No ns >0.9999 E2:+/+ vs. E2:-/- 0.61 0.3633 to 0.8568 Yes **** <0.0001 E2:+/+ vs. E2:+/+;+/- 0.07524 -0.1487 to 0.2992 No ns 0.9980 E2:+/+ vs. E2:+/+;-,- 1.074 0.8438 to 1.303 Yes **** <0.0001 E2:+/+ vs. E2:-/-;-/- 1.032 0.8023 to 1.262 Yes **** <0.0001 E2:+/+ vs. G-1:+/+ 0.02702 -0.2197 to 0.2738 No ns >0.9999 E2:+/+ vs. G-1:-/- 0.6434 0.4219 to 0.8649 Yes **** <0.0001 E2:+/+ vs. G-1:+/+;+/- 0.2539 0.04016 to 0.4676 Yes ** 0.0056 E2:+/+ vs. G-1:+/+;-,- 1.057 0.8203 to 1.294 Yes **** <0.0001 E2:+/+ vs. G-1:-/-;-/- 1.064 0.8114 to 1.317 Yes **** <0.0001 E2:-/- vs. E2:+/+;+/- -0.5348 -0.7587 to -0.3109 Yes **** <0.0001 E2:-/- vs. E2:+/+;-,- 0.4635 0.2338 to 0.6932 Yes **** <0.0001 E2:-/- vs. E2:-/-;-/- 0.422 0.1923 to 0.6518 Yes **** <0.0001 E2:-/- vs. G-1:+/+ -0.583 -0.8298 to -0.3363 Yes **** <0.0001 E2:-/- vs. G-1:-/- 0.03338 -0.1881 to 0.2549 No ns >0.9999 E2:-/- vs. G-1:+/+;+/- -0.3562 -0.5699 to -0.1425 Yes **** <0.0001 E2:-/- vs. G-1:+/+;-,- 0.4474 0.2103 to 0.6844 Yes **** <0.0001 E2:-/- vs. G-1:-/-;-/- 0.4542 0.2014 to 0.7071 Yes **** <0.0001 E2:+/+;+/- vs. E2:+/+;-,- 0.9983 0.7933 to 1.203 Yes **** <0.0001 E2:+/+;+/- vs. E2:-/-;-/- 0.9568 0.7518 to 1.162 Yes **** <0.0001 E2:+/+;+/- vs. G-1:+/+ -0.04823 -0.2722 to 0.1757 No ns >0.9999 E2:+/+;+/- vs. G-1:-/- 0.5682 0.3725 to 0.7639 Yes **** <0.0001 E2:+/+;+/- vs. G-1:+/+;+/- 0.1786 -0.008261 to 0.3655 No ns 0.0778 E2:+/+;+/- vs. G-1:+/+;-,- 0.9822 0.7689 to 1.195 Yes **** <0.0001 E2:+/+;+/- vs. G-1:-/-;-/- 0.989 0.7584 to 1.22 Yes **** <0.0001 E2:+/+;-,- vs. E2:-/-;-/- -0.04147 -0.2528 to 0.1698 No ns >0.9999 E2:+/+;-,- vs. G-1:+/+ -1.047 -1.276 to -0.8168 Yes **** <0.0001 E2:+/+;-,- vs. G-1:-/- -0.4301 -0.6324 to -0.2278 Yes **** <0.0001 E2:+/+;-,- vs. G-1:+/+;+/- -0.8197 -1.013 to -0.6259 Yes **** <0.0001 E2:+/+;-,- vs. G-1:+/+;-,- -0.01614 -0.2354 to 0.2031 No ns >0.9999 E2:+/+;-,- vs. G-1:-/-;-/- -0.009285 -0.2455 to 0.227 No ns >0.9999 E2:-/-;-/- vs. G-1:+/+ -1.005 -1.235 to -0.7753 Yes **** <0.0001 E2:-/-;-/- vs. G-1:-/- -0.3887 -0.591 to -0.1863 Yes **** <0.0001 E2:-/-;-/- vs. G-1:+/+;+/- -0.7782 -0.972 to -0.5844 Yes **** <0.0001 E2:-/-;-/- vs. G-1:+/+;-,- 0.02533 -0.194 to 0.2446 No ns >0.9999 E2:-/-;-/- vs. G-1:-/-;-/- 0.03218 -0.2041 to 0.2684 No ns >0.9999 G-1:+/+ vs. G-1:-/- 0.6164 0.3949 to 0.8379 Yes **** <0.0001 G-1:+/+ vs. G-1:+/+;+/- 0.2268 0.01314 to 0.4405 Yes * 0.0258 G-1:+/+ vs. G-1:+/+;-,- 1.03 0.7933 to 1.267 Yes **** <0.0001 G-1:+/+ vs. G-1:-/-;-/- 1.037 0.7844 to 1.29 Yes **** <0.0001 G-1:-/- vs. G-1:+/+;+/- -0.3896 -0.5735 to -0.2056 Yes **** <0.0001 G-1:-/- vs. G-1:+/+;-,- 0.414 0.2033 to 0.6246 Yes **** <0.0001 G-1:-/- vs. G-1:-/-;-/- 0.4208 0.1926 to 0.6491 Yes **** <0.0001 G-1:+/+;+/- vs. G-1:+/+;-,- 0.8035 0.6011 to 1.006 Yes **** <0.0001 G-1:+/+;+/- vs. G-1:-/-;-/- 0.8104 0.5897 to 1.031 Yes **** <0.0001 G-1:+/+;-,- vs. G-1:-/-;-/- 0.006855 -0.2366 to 0.2503 No ns >0.9999

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Interaction 4.244 <0.0001 **** Yes Treatment Factor 1.905 0.0010 ** Yes Genetic Factor 69.46 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.1113 6 0.01854 F (6, 185) = 5.303 P<0.0001 Treatment Factor 0.04994 2 0.02497 F (2, 185) = 7.141 P=0.0010 Genetic Factor 1.821 3 0.6069 F (3, 185) = 173.6 P<0.0001 Residual 0.6469 185 0.003497

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ ; mtor +/+ vs. DMSO:gper1 -/- ; mtor +/+ 0.09214 0.03008 to 0.1542 Yes *** 0.0001

DMSO:gper1 +/+ ; mtor +/+ vs. DMSO:gper1 +/+ ; mtor +/- 0.05953 -0.003083 to 0.1221 No ns 0.0793

DMSO:gper1 +/+ ; mtor +/+ vs. DMSO:gper1 -/- ; mtor -/- 0.2281 0.1543 to 0.3019 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/+ -0.08155 -0.1487 to -0.0144 Yes ** 0.0047

DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor +/+ 0.1088 0.04677 to 0.1709 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- -0.00843 -0.08418 to 0.06732 No ns >0.9999

DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.23 0.146 to 0.3139 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.1223 -0.1961 to -0.04849 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ 0.08963 0.02642 to 0.1528 Yes *** 0.0003

DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- 0.03514 -0.03866 to 0.109 No ns 0.9155

DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.2356 0.1662 to 0.3049 Yes **** <0.0001

DMSO:gper1 -/- ; mtor +/+ vs. DMSO:gper1 +/+ ; mtor +/- -0.03261 -0.08973 to 0.02451 No ns 0.7641

DMSO:gper1 -/- ; mtor +/+ vs. DMSO:gper1 -/- ; mtor -/- 0.136 0.06677 to 0.2052 Yes **** <0.0001

DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/+ -0.1737 -0.2357 to -0.1116 Yes **** <0.0001

DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 -/- ; mtor +/+ 0.01669 -0.03982 to 0.0732 No ns 0.998

DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- -0.1006 -0.1718 to -0.02929 Yes *** 0.0004

DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.1378 0.05791 to 0.2177 Yes **** <0.0001

DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.2144 -0.2836 to -0.1452 Yes **** <0.0001

DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ -0.002503 -0.06028 to 0.05528 No ns >0.9999

DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- -0.05699 -0.1262 to 0.01222 No ns 0.2221

DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.1435 0.07903 to 0.2079 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/- vs. DMSO:gper1 -/- ; mtor -/- 0.1686 0.09888 to 0.2383 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 +/+ ; mtor +/+ -0.1411 -0.2037 to -0.07846 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 -/- ; mtor +/+ 0.0493 -0.007823 to 0.1064 No ns 0.1656

DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 +/+ ; mtor +/- -0.06796 -0.1397 to 0.003803 No ns 0.0821

DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 -/- ; mtor -/- 0.1704 0.09008 to 0.2508 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/+ -0.1818 -0.2515 to -0.1121 Yes **** <0.0001

DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor +/+ 0.0301 -0.02827 to 0.08848 No ns 0.8629

DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/- -0.02438 -0.0941 to 0.04533 No ns 0.9913

DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor -/- 0.1761 0.1111 to 0.241 Yes **** <0.0001

DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 +/+ ; mtor +/+ -0.3097 -0.3835 to -0.2359 Yes **** <0.0001

DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 -/- ; mtor +/+ -0.1193 -0.1885 to -0.05008 Yes **** <0.0001

DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 +/+ ; mtor +/- -0.2365 -0.3183 to -0.1548 Yes **** <0.0001

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DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 -/- ; mtor -/- 0.001846 -0.0875 to 0.0912 No ns >0.9999

DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/+ -0.3504 -0.4303 to -0.2705 Yes **** <0.0001

DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor +/+ -0.1385 -0.2087 to -0.06823 Yes **** <0.0001

DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/- -0.193 -0.2729 to -0.1131 Yes **** <0.0001

DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor -/- 0.007476 -0.06834 to 0.08329 No ns >0.9999

E2:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor +/+ 0.1904 0.1283 to 0.2524 Yes **** <0.0001

E2:gper1 +/+ ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- 0.07312 -0.002633 to 0.1489 No ns 0.0695

E2:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.3115 0.2276 to 0.3954 Yes **** <0.0001

E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.04075 -0.1146 to 0.03306 No ns 0.8007

E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ 0.1712 0.108 to 0.2344 Yes **** <0.0001

E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- 0.1167 0.04288 to 0.1905 Yes **** <0.0001

E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.3171 0.2478 to 0.3865 Yes **** <0.0001

E2:gper1 -/- ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- -0.1173 -0.1885 to -0.04598 Yes **** <0.0001

E2:gper1 -/- ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.1211 0.04122 to 0.2011 Yes **** <0.0001

E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.2311 -0.3003 to -0.1619 Yes **** <0.0001

E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ -0.01919 -0.07697 to 0.03859 No ns 0.9944

E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- -0.07368 -0.1429 to -0.004472 Yes * 0.026

E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.1268 0.06234 to 0.1912 Yes **** <0.0001

E2:gper1 +/+ ; mtor +/- vs. E2:gper1 -/- ; mtor -/- 0.2384 0.1474 to 0.3294 Yes **** <0.0001

E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/+ -0.1139 -0.1956 to -0.03215 Yes *** 0.0004

E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor +/+ 0.09806 0.02578 to 0.1704 Yes *** 0.0007

E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/- 0.04357 -0.03814 to 0.1253 No ns 0.8344

E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor -/- 0.244 0.1663 to 0.3217 Yes **** <0.0001

E2:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/+ -0.3523 -0.4416 to -0.2629 Yes **** <0.0001

E2:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor +/+ -0.1403 -0.2212 to -0.05951 Yes **** <0.0001

E2:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/- -0.1948 -0.2842 to -0.1055 Yes **** <0.0001

E2:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor -/- 0.00563 -0.08007 to 0.09133 No ns >0.9999

G-1:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ 0.2119 0.1417 to 0.2822 Yes **** <0.0001

G-1:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- 0.1574 0.07752 to 0.2374 Yes **** <0.0001

G-1:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.3579 0.2821 to 0.4337 Yes **** <0.0001

G-1:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- -0.05449 -0.1247 to 0.01576 No ns 0.3059

G-1:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.146 0.08041 to 0.2115 Yes **** <0.0001

G-1:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor -/- 0.2005 0.1246 to 0.2763 Yes **** <0.0001

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Figure 5E:

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.613 0.2150 ns No Mtz Factor 0.01651 0.8581 ns No Genetic Factor 58.51 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1122 2 561.1 F (2, 68) = 1.572 P=0.2150 Mtz Factor 11.49 1 11.49 F (1, 68) = 0.03219 P=0.8581 Genetic Factor 40711 2 20356 F (2, 68) = 57.04 P<0.0001 Residual 24269 68 356.9

Sidak's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

No MTz:gper1 +/+ vs. No MTz:gper1 -/- 12.37 -11.26 to 36 No ns 0.845

No MTz:gper1 +/+ vs. No MTz:ztor MUT 50.54 28.92 to 72.15 Yes **** <0.0001

No MTz:gper1 +/+ vs. MTz:gper1 +/+ -11.11 -29.44 to 7.229 No ns 0.6657

No MTz:gper1 +/+ vs. MTz:gper1 -/- 13.27 -12.47 to 39.01 No ns 0.8589

No MTz:gper1 +/+ vs. MTz:ztor MUT 58.1 30.88 to 85.32 Yes **** <0.0001

No MTz:gper1 -/- vs. No MTz:ztor MUT 38.17 12.89 to 63.44 Yes *** 0.0003

No MTz:gper1 -/- vs. MTz:gper1 +/+ -23.47 -46.01 to -0.9364 Yes * 0.0347

No MTz:gper1 -/- vs. MTz:gper1 -/- 0.9026 -27.99 to 29.79 No ns >0.9999

No MTz:gper1 -/- vs. MTz:ztor MUT 45.73 15.52 to 75.94 Yes *** 0.0003

No MTz:ztor MUT vs. MTz:gper1 +/+ -61.64 -82.06 to -41.23 Yes **** <0.0001

No MTz:ztor MUT vs. MTz:gper1 -/- -37.27 -64.53 to -10 Yes ** 0.0014No MTz:ztor MUT vs. MTz:ztor MUT 7.564 -21.1 to 36.22 No ns 0.9998

MTz:gper1 +/+ vs. MTz:gper1 -/- 24.38 -0.3672 to 49.12 No ns 0.0568

MTz:gper1 +/+ vs. MTz:ztor MUT 69.21 42.93 to 95.48 Yes **** <0.0001

MTz:gper1 -/- vs. MTz:ztor MUT 44.83 12.94 to 76.72 Yes *** 0.0009

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Figure 6B:

Male

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 8.354 0.0005 *** Yes GPER1 Factor 68.38 <0.0001 **** Yes E2 Treatment Factor 6.569 0.0018 ** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 5.025 1 5.025 F (1, 36) = 14.4 P=0.0005 GPER1 Factor 41.13 1 41.13 F (1, 36) = 117.9 P<0.0001 E2 Treatment Factor 3.951 1 3.951 F (1, 36) = 11.32 P=0.0018 Residual 12.56 36 0.3489

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- -0.08048 -0.7919 to 0.631 No ns 0.99

DMSO:gper1 +/+ vs. E2:gper1 +/+ -2.744 -3.475 to -2.013 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1 -/- -1.403 -2.098 to -0.7078 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 +/+ -2.663 -3.394 to -1.932 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 -/- -1.322 -2.018 to -0.6274 Yes **** <0.0001

E2:gper1 +/+ vs. E2:gper1 -/- 1.341 0.6258 to 2.056 Yes **** <0.0001

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Figure 6B:

Female

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 0.1474 0.7367 ns No GPER1 Factor 56.59 <0.0001 **** Yes E2 Treatment Factor 5.063 0.0566 ns No ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.03853 1 0.03853 F (1, 27) = 0.1154 P=0.7367 GPER1 Factor 14.79 1 14.79 F (1, 27) = 44.33 P<0.0001 E2 Treatment Factor 1.324 1 1.324 F (1, 27) = 3.966 P=0.0566 Residual 9.011 27 0.3337

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.4994 -0.2505 to 1.249 No ns 0.2851

DMSO:gper1 +/+ vs. E2:gper1 +/+ -1.353 -2.103 to -0.6036 Yes *** 0.0002

DMSO:gper1 +/+ vs. E2:gper1 -/- -0.9997 -1.866 to -0.1338 Yes * 0.0191

DMSO:gper1 -/- vs. E2:gper1 +/+ -1.853 -2.643 to -1.062 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 -/- -1.499 -2.4 to -0.5978 Yes *** 0.0006

E2:gper1 +/+ vs. E2:gper1 -/- 0.3538 -0.5474 to 1.255 No ns 0.7077

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Figure 7H:

One-way ANOVA

Male Number of families 1 Number of comparisons per family 6 Alpha 0.05 Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value DMSO vs. E2 -0.7606 -1.42 to -0.1013 Yes * 0.0172 DMSO vs. E2 +G15 -0.08686 -0.7634 to 0.5897 No ns 0.9867 DMSO vs. E2 +Ful -2.092 -3.086 to -1.097 Yes **** <0.0001 E2 vs. E2 +G15 0.6737 -0.03891 to 1.386 No ns 0.0707 E2 vs. E2 +Ful -1.331 -2.35 to -0.3119 Yes ** 0.0053 E2 +G15 vs. E2 +Ful -2.005 -3.035 to -0.9743 Yes **** <0.0001 Female

Number of families 1 Number of comparisons per family 6 Alpha 0.05 Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value DMSO vs. E2 -0.045 -1.011 to 0.9207 No ns 0.9993 DMSO vs. E2 +G15 0.3333 -0.9571 to 1.624 No ns 0.9032 DMSO vs. E2 +Ful -0.05705 -1.189 to 1.075 No ns 0.9991 E2 vs. E2 +G15 0.3783 -0.7466 to 1.503 No ns 0.8105 E2 vs. E2 +Ful -0.01205 -0.9508 to 0.9267 No ns >0.9999 E2 +G15 vs. E2 +Ful -0.3904 -1.661 to 0.88 No ns 0.8484

Figure S2C:

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Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 5.219 <0.0001 **** Yes Treatment E2 Factor 34.16 <0.0001 **** Yes MOs Factor 20.17 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1.312 5 0.2624 F (5, 240) = 6.302 P<0.0001 Treatment E2 Factor 8.585 1 8.585 F (1, 240) = 206.2 P<0.0001 MOs Factor 5.07 5 1.014 F (5, 240) = 24.36 P<0.0001 Residual 9.991 240 0.04163

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P ValueDMSO:con vs. DMSO:esr1 -0.03659 -0.2231 to 0.1499 No ns >0.9999DMSO:con vs. DMSO:esr2a 0.1031 -0.08346 to 0.2896 No ns 0.8034DMSO:con vs. DMSO:esr2b 0.1161 -0.073 to 0.3053 No ns 0.6748DMSO:con vs. DMSO:esr2a+2b -0.04011 -0.2681 to 0.1879 No ns >0.9999DMSO:con vs. DMSO:gper1 0.1945 -0.003962 to 0.3929 No ns 0.0606DMSO:con vs. E2:con -0.4567 -0.6264 to -0.2869 Yes **** <0.0001DMSO:con vs. E2:esr1 -0.3783 -0.5845 to -0.1721 Yes **** <0.0001DMSO:con vs. E2:esr2a -0.3783 -0.5845 to -0.1721 Yes **** <0.0001DMSO:con vs. E2:esr2b -0.3375 -0.5359 to -0.1391 Yes **** <0.0001DMSO:con vs. E2:esr2a+2b -0.5442 -0.7657 to -0.3227 Yes **** <0.0001DMSO:con vs. E2:gper1 0.1051 -0.06462 to 0.2748 No ns 0.6628DMSO:esr1 vs. DMSO:esr2a 0.1396 -0.06341 to 0.3427 No ns 0.5009DMSO:esr1 vs. DMSO:esr2b 0.1527 -0.05274 to 0.3582 No ns 0.3748DMSO:esr1 vs. DMSO:esr2a+2b -0.003522 -0.2452 to 0.2382 No ns >0.9999DMSO:esr1 vs. DMSO:gper1 0.231 0.01701 to 0.4451 Yes * 0.022DMSO:esr1 vs. E2:con -0.4201 -0.6078 to -0.2323 Yes **** <0.0001DMSO:esr1 vs. E2:esr1 -0.3417 -0.563 to -0.1205 Yes **** <0.0001DMSO:esr1 vs. E2:esr2a -0.3417 -0.563 to -0.1205 Yes **** <0.0001DMSO:esr1 vs. E2:esr2b -0.3009 -0.5149 to -0.08686 Yes *** 0.0004DMSO:esr1 vs. E2:esr2a+2b -0.5076 -0.7432 to -0.272 Yes **** <0.0001DMSO:esr1 vs. E2:gper1 0.1417 -0.04605 to 0.3294 No ns 0.3507DMSO:esr2a vs. DMSO:esr2b 0.01308 -0.1924 to 0.2185 No ns >0.9999DMSO:esr2a vs. DMSO:esr2a+2b -0.1432 -0.3849 to 0.09852 No ns 0.7229DMSO:esr2a vs. DMSO:gper1 0.0914 -0.1226 to 0.3054 No ns 0.9608DMSO:esr2a vs. E2:con -0.5597 -0.7474 to -0.372 Yes **** <0.0001DMSO:esr2a vs. E2:esr1 -0.4814 -0.7027 to -0.2601 Yes **** <0.0001DMSO:esr2a vs. E2:esr2a -0.4814 -0.7027 to -0.2601 Yes **** <0.0001DMSO:esr2a vs. E2:esr2b -0.4405 -0.6546 to -0.2265 Yes **** <0.0001DMSO:esr2a vs. E2:esr2a+2b -0.6473 -0.8829 to -0.4117 Yes **** <0.0001DMSO:esr2a vs. E2:gper1 0.002041 -0.1857 to 0.1898 No ns >0.9999DMSO:esr2b vs. DMSO:esr2a+2b -0.1562 -0.4 to 0.08746 No ns 0.6118DMSO:esr2b vs. DMSO:gper1 0.07832 -0.138 to 0.2946 No ns 0.9889

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DMSO:esr2b vs. E2:con -0.5728 -0.7631 to -0.3824 Yes **** <0.0001DMSO:esr2b vs. E2:esr1 -0.4945 -0.7179 to -0.271 Yes **** <0.0001DMSO:esr2b vs. E2:esr2a -0.4945 -0.7179 to -0.271 Yes **** <0.0001DMSO:esr2b vs. E2:esr2b -0.4536 -0.6699 to -0.2373 Yes **** <0.0001DMSO:esr2b vs. E2:esr2a+2b -0.6603 -0.898 to -0.4227 Yes **** <0.0001DMSO:esr2b vs. E2:gper1 -0.01104 -0.2014 to 0.1793 No ns >0.9999DMSO:esr2a+2b vs. DMSO:gper1 0.2346 -0.01642 to 0.4856 No ns 0.092DMSO:esr2a+2b vs. E2:con -0.4165 -0.6455 to -0.1876 Yes **** <0.0001DMSO:esr2a+2b vs. E2:esr1 -0.3382 -0.5954 to -0.08104 Yes ** 0.0012DMSO:esr2a+2b vs. E2:esr2a -0.3382 -0.5954 to -0.08104 Yes ** 0.0012DMSO:esr2a+2b vs. E2:esr2b -0.2974 -0.5484 to -0.04639 Yes ** 0.0066DMSO:esr2a+2b vs. E2:esr2a+2b -0.5041 -0.7737 to -0.2345 Yes **** <0.0001DMSO:esr2a+2b vs. E2:gper1 0.1452 -0.08376 to 0.3742 No ns 0.6281DMSO:gper1 vs. E2:con -0.6511 -0.8507 to -0.4515 Yes **** <0.0001DMSO:gper1 vs. E2:esr1 -0.5728 -0.8042 to -0.3414 Yes **** <0.0001DMSO:gper1 vs. E2:esr2a -0.5728 -0.8042 to -0.3414 Yes **** <0.0001DMSO:gper1 vs. E2:esr2b -0.5319 -0.7564 to -0.3075 Yes **** <0.0001DMSO:gper1 vs. E2:esr2a+2b -0.7387 -0.9838 to -0.4935 Yes **** <0.0001DMSO:gper1 vs. E2:gper1 -0.08936 -0.2889 to 0.1102 No ns 0.9453E2:con vs. E2:esr1 0.07832 -0.129 to 0.2856 No ns 0.9844E2:con vs. E2:esr2a 0.07832 -0.129 to 0.2856 No ns 0.9844E2:con vs. E2:esr2b 0.1192 -0.0804 to 0.3187 No ns 0.7126E2:con vs. E2:esr2a+2b -0.08756 -0.3101 to 0.135 No ns 0.9786E2:con vs. E2:gper1 0.5617 0.3907 to 0.7328 Yes **** <0.0001E2:esr1 vs. E2:esr2a 0 -0.2381 to 0.2381 No ns >0.9999E2:esr1 vs. E2:esr2b 0.04084 -0.1906 to 0.2722 No ns >0.9999E2:esr1 vs. E2:esr2a+2b -0.1659 -0.4173 to 0.08558 No ns 0.5677E2:esr1 vs. E2:gper1 0.4834 0.2761 to 0.6907 Yes **** <0.0001E2:esr2a vs. E2:esr2b 0.04084 -0.1906 to 0.2722 No ns >0.9999E2:esr2a vs. E2:esr2a+2b -0.1659 -0.4173 to 0.08558 No ns 0.5677E2:esr2a vs. E2:gper1 0.4834 0.2761 to 0.6907 Yes **** <0.0001E2:esr2b vs. E2:esr2a+2b -0.2067 -0.4518 to 0.0384 No ns 0.1938E2:esr2b vs. E2:gper1 0.4426 0.243 to 0.6422 Yes **** <0.0001E2:esr2a+2b vs. E2:gper1 0.6493 0.4268 to 0.8718 Yes **** <0.0001

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Figure S5F:

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 20.16 <0.0001 **** Yes GPER1 Factor 30.16 <0.0001 **** Yes Treatments Factor 15.09 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 3.751 5 0.7502 F (5, 170) = 26.43 P<0.0001 GPER1 Factor 5.613 5 1.123 F (5, 170) = 39.55 P<0.0001 Treatments Factor 2.809 1 2.809 F (1, 170) = 98.98 P<0.0001 Residual 4.825 170 0.02838

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1-/- 0.1459 -0.03293 to 0.3248 No ns 0.2335

DMSO:gper1 +/+ vs. E2:gper1 +/+ -0.6312 -0.8123 to -0.45 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1-/- 0.01198 -0.1847 to 0.2086 No ns >0.9999

DMSO:gper1 +/+ vs. G1:gper1 +/+ -0.6046 -0.8104 to -0.3987 Yes **** <0.0001

DMSO:gper1 +/+ vs. G1:gper1-/- 0.1179 -0.1002 to 0.3361 No ns 0.8202

DMSO:gper1 +/+ vs. Rapa:gper1 +/+ 0.1946 0.008174 to 0.381 Yes * 0.0323

DMSO:gper1 +/+ vs. Rapa:gper1-/- 0.1042 -0.1311 to 0.3395 No ns 0.9473

DMSO:gper1 +/+ vs. Rapa+E2:gper1 +/+ 0.004425 -0.1965 to 0.2054 No ns >0.9999

DMSO:gper1 +/+ vs. Rapa+E2:gper1-/- 0.0678 -0.1217 to 0.2572 No ns 0.9894

DMSO:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.09971 -0.08669 to 0.2861 No ns 0.8303

DMSO:gper1 +/+ vs. Rapa+G1:gper1-/- 0.1612 -0.02517 to 0.3476 No ns 0.1627

DMSO:gper1-/- vs. E2:gper1 +/+ -0.7771 -0.956 to -0.5982 Yes **** <0.0001DMSO:gper1-/- vs. E2:gper1-/- -0.134 -0.3285 to 0.0606 No ns 0.4935

DMSO:gper1-/- vs. G1:gper1 +/+ -0.7505 -0.9544 to -0.5466 Yes **** <0.0001DMSO:gper1-/- vs. G1:gper1-/- -0.028 -0.2442 to 0.1882 No ns >0.9999

DMSO:gper1-/- vs. Rapa:gper1 +/+ 0.04863 -0.1356 to 0.2328 No ns 0.9993DMSO:gper1-/- vs. Rapa:gper1-/- -0.04177 -0.2753 to 0.1918 No ns >0.9999

DMSO:gper1-/- vs. Rapa+E2:gper1 +/+ -0.1415 -0.3404 to 0.0574 No ns 0.4405DMSO:gper1-/- vs. Rapa+E2:gper1-/- -0.07814 -0.2654 to 0.1091 No ns 0.9653

DMSO:gper1-/- vs. Rapa+G1:gper1 +/+ -0.04623 -0.2304 to 0.138 No ns 0.9995DMSO:gper1-/- vs. Rapa+G1:gper1-/- 0.01529 -0.1689 to 0.1995 No ns >0.9999

E2:gper1 +/+ vs. E2:gper1-/- 0.6431 0.4465 to 0.8398 Yes **** <0.0001

E2:gper1 +/+ vs. G1:gper1 +/+ 0.02659 -0.1793 to 0.2325 No ns >0.9999

E2:gper1 +/+ vs. G1:gper1-/- 0.7491 0.531 to 0.9672 Yes **** <0.0001

E2:gper1 +/+ vs. Rapa:gper1 +/+ 0.8257 0.6393 to 1.012 Yes **** <0.0001

E2:gper1 +/+ vs. Rapa:gper1-/- 0.7353 0.5 to 0.9706 Yes **** <0.0001

E2:gper1 +/+ vs. Rapa+E2:gper1 +/+ 0.6356 0.4346 to 0.8365 Yes **** <0.0001

E2:gper1 +/+ vs. Rapa+E2:gper1-/- 0.699 0.5095 to 0.8884 Yes **** <0.0001

E2:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.7309 0.5445 to 0.9173 Yes **** <0.0001

E2:gper1 +/+ vs. Rapa+G1:gper1-/- 0.7924 0.606 to 0.9788 Yes **** <0.0001

E2:gper1-/- vs. G1:gper1 +/+ -0.6165 -0.8362 to -0.3969 Yes **** <0.0001E2:gper1-/- vs. G1:gper1-/- 0.106 -0.1252 to 0.3371 No ns 0.9334

E2:gper1-/- vs. Rapa:gper1 +/+ 0.1826 -0.01891 to 0.3841 No ns 0.1168E2:gper1-/- vs. Rapa:gper1-/- 0.09219 -0.1553 to 0.3396 No ns 0.9854

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E2:gper1-/- vs. Rapa+E2:gper1 +/+ -0.007554 -0.2226 to 0.2075 No ns >0.9999E2:gper1-/- vs. Rapa+E2:gper1-/- 0.05582 -0.1485 to 0.2601 No ns 0.999

E2:gper1-/- vs. Rapa+G1:gper1 +/+ 0.08773 -0.1138 to 0.2892 No ns 0.9531E2:gper1-/- vs. Rapa+G1:gper1-/- 0.1493 -0.05225 to 0.3508 No ns 0.3758

G1:gper1 +/+ vs. G1:gper1-/- 0.7225 0.4834 to 0.9616 Yes **** <0.0001

G1:gper1 +/+ vs. Rapa:gper1 +/+ 0.7991 0.5886 to 1.01 Yes **** <0.0001

G1:gper1 +/+ vs. Rapa:gper1-/- 0.7087 0.4539 to 0.9636 Yes **** <0.0001

G1:gper1 +/+ vs. Rapa+E2:gper1 +/+ 0.609 0.3855 to 0.8325 Yes **** <0.0001

G1:gper1 +/+ vs. Rapa+E2:gper1-/- 0.6724 0.4591 to 0.8856 Yes **** <0.0001

G1:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.7043 0.4938 to 0.9148 Yes **** <0.0001

G1:gper1 +/+ vs. Rapa+G1:gper1-/- 0.7658 0.5553 to 0.9763 Yes **** <0.0001

G1:gper1-/- vs. Rapa:gper1 +/+ 0.07663 -0.1459 to 0.2991 No ns 0.9923G1:gper1-/- vs. Rapa:gper1-/- -0.01377 -0.2786 to 0.2511 No ns >0.9999

G1:gper1-/- vs. Rapa+E2:gper1 +/+ -0.1135 -0.3484 to 0.1213 No ns 0.9062G1:gper1-/- vs. Rapa+E2:gper1-/- -0.05014 -0.2752 to 0.1749 No ns 0.9999

G1:gper1-/- vs. Rapa+G1:gper1 +/+ -0.01823 -0.2407 to 0.2043 No ns >0.9999G1:gper1-/- vs. Rapa+G1:gper1-/- 0.04329 -0.1792 to 0.2658 No ns >0.9999

Rapa:gper1 +/+ vs. Rapa:gper1-/- -0.0904 -0.3298 to 0.149 No ns 0.9837

Rapa:gper1 +/+ vs. Rapa+E2:gper1 +/+ -0.1901 -0.3959 to 0.01556 No ns 0.1

Rapa:gper1 +/+ vs. Rapa+E2:gper1-/- -0.1268 -0.3212 to 0.0677 No ns 0.5804

Rapa:gper1 +/+ vs. Rapa+G1:gper1 +/+ -0.09486 -0.2864 to 0.09664 No ns 0.891

Rapa:gper1 +/+ vs. Rapa+G1:gper1-/- -0.03334 -0.2248 to 0.1582 No ns >0.9999

Rapa:gper1-/- vs. Rapa+E2:gper1 +/+ -0.09974 -0.3506 to 0.1511 No ns 0.9758Rapa:gper1-/- vs. Rapa+E2:gper1-/- -0.03637 -0.2781 to 0.2054 No ns >0.9999

Rapa:gper1-/- vs. Rapa+G1:gper1 +/+ -0.004459 -0.2438 to 0.2349 No ns >0.9999Rapa:gper1-/- vs. Rapa+G1:gper1-/- 0.05706 -0.1823 to 0.2964 No ns 0.9997

Rapa+E2:gper1 +/+ vs. Rapa+E2:gper1-/- 0.06337 -0.1451 to 0.2718 No ns 0.9974

Rapa+E2:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.09529 -0.1104 to 0.301 No ns 0.9286

Rapa+E2:gper1 +/+ vs. Rapa+G1:gper1-/- 0.1568 -0.0489 to 0.3625 No ns 0.3311

Rapa+E2:gper1-/- vs. Rapa+G1:gper1 +/+ 0.03191 -0.1626 to 0.2264 No ns >0.9999Rapa+E2:gper1-/- vs. Rapa+G1:gper1-/- 0.09343 -0.101 to 0.2879 No ns 0.9097

Rapa+G1:gper1 +/+ vs. Rapa+G1:gper1-/- 0.06152 -0.13 to 0.253 No ns 0.9957

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Figure S6A: Male

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 18.47 <0.0001 **** Yes GPER1 Factor 51.16 <0.0001 **** Yes E2 Treatment Factor 22.5 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 223.4 1 223.4 F (1, 30) = 39.61 P<0.0001 GPER1 Factor 618.8 1 618.8 F (1, 30) = 109.7 P<0.0001 E2 Treatment Factor 272.1 1 272.1 F (1, 30) = 48.25 P<0.0001 Residual 169.2 30 5.64

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.54 -2.348 to 3.428 No ns 0.9564

DMSO:gper1 +/+ vs. E2:gper1 +/+ -13.88 -17.06 to -10.69 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1 -/- -2.92 -6.102 to 0.2623 No ns 0.0812

DMSO:gper1 -/- vs. E2:gper1 +/+ -14.42 -17.6 to -11.23 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 -/- -3.46 -6.642 to -0.2777 Yes * 0.029

E2:gper1 +/+ vs. E2:gper1 -/- 10.96 7.505 to 14.41 Yes **** <0.0001

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Figure S6A: Female

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 0.2994 0.6605 ns No GPER1 Factor 29.41 0.0001 *** Yes E2 Treatment Factor 28.21 0.0002 *** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 4.86 1 4.86 F (1, 30) = 0.1968 P=0.6605 GPER1 Factor 477.3 1 477.3 F (1, 30) = 19.33 P=0.0001 E2 Treatment Factor 457.9 1 457.9 F (1, 30) = 18.54 P=0.0002 Residual 741 30 24.7

Figure S6B:

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 6.642 0.4332 to 12.85 Yes * 0.0325

DMSO:gper1 +/+ vs. E2:gper1 +/+ -8.323 -14.98 to -1.663 Yes ** 0.0099

DMSO:gper1 +/+ vs. E2:gper1 -/- -0.155 -6.565 to 6.255 No ns 0.9999

DMSO:gper1 -/- vs. E2:gper1 +/+ -14.97 -21.78 to -8.155 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 -/- -6.797 -13.36 to -0.2309 Yes * 0.0403

E2:gper1 +/+ vs. E2:gper1 -/- 8.168 1.174 to 15.16 Yes * 0.0172

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Male

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 8.594 0.0218 * Yes GPER1 Factor 1.425 0.3325 ns No E2 Treatment Factor 51.65 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 15952 1 15952 F (1, 30) = 5.851 P=0.0218 GPER1 Factor 2646 1 2646 F (1, 30) = 0.9704 P=0.3325 E2 Treatment Factor 95882 1 95882 F (1, 30) = 35.17 P<0.0001 Residual 81793 30 2726

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary ted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 63.89 0.3949 to 127.4 Yes * 0.0481

DMSO:gper1 +/+ vs. E2:gper1 +/+ -26.09 -96.06 to 43.88 No ns 0.7427

DMSO:gper1 +/+ vs. E2:gper1 -/- 125.8 55.86 to 195.8 Yes *** 0.0002

DMSO:gper1 -/- vs. E2:gper1 +/+ -89.98 -159.9 to -20.01 Yes ** 0.0077

DMSO:gper1 -/- vs. E2:gper1 -/- 61.94 -8.032 to 131.9 No ns 0.0974

E2:gper1 +/+ vs. E2:gper1 -/- 151.9 76.02 to 227.8 Yes **** <0.0001

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Figure S6B: Female

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.526 0.2568 ns No GPER1 Factor 16.86 0.0006 *** Yes E2 Treatment Factor 41.54 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 5242 1 5242 F (1, 30) = 1.337 P=0.2568 GPER1 Factor 57923 1 57923 F (1, 30) = 14.77 P=0.0006 E2 Treatment Factor 142701 1 142701 F (1, 30) = 36.38 P<0.0001 Residual 117666 30 3922

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary ted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 155.8 77.53 to 234 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1 +/+ 108.3 24.42 to 192.3 Yes ** 0.0074

DMSO:gper1 +/+ vs. E2:gper1 -/- 214 133.2 to 294.8 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 +/+ -47.44 -133.3 to 38.38 No ns 0.4483

DMSO:gper1 -/- vs. E2:gper1 -/- 58.23 -24.52 to 141 No ns 0.244

E2:gper1 +/+ vs. E2:gper1 -/- 105.7 17.53 to 193.8 Yes * 0.014

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Figure S6C: Male

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 2.14 0.2338 ns No GPER1 Factor 14.49 0.0036 ** Yes E2 Treatment Factor 35.5 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.04501 1 0.04501 F (1, 30) = 1.476 P=0.2338 GPER1 Factor 0.3047 1 0.3047 F (1, 30) = 9.995 P=0.0036 E2 Treatment Factor 0.7465 1 0.7465 F (1, 30) = 24.48 P<0.0001 Residual 0.9146 30 0.03049

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.375 0.1627 to 0.5873 Yes *** 0.0002

DMSO:gper1 +/+ vs. E2:gper1 +/+ 0.2663 0.03231 to 0.5003 Yes * 0.0209

DMSO:gper1 +/+ vs. E2:gper1 -/- 0.4934 0.2595 to 0.7274 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 +/+ -0.1087 -0.3427 to 0.1253 No ns 0.5926

DMSO:gper1 -/- vs. E2:gper1 -/- 0.1184 -0.1155 to 0.3524 No ns 0.5234

E2:gper1 +/+ vs. E2:gper1 -/- 0.2271 -0.02664 to 0.4809 No ns 0.0922

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Figure S6C: Female

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 3.482 0.0251 * Yes GPER1 Factor 9.218 0.0006 *** Yes E2 Treatment Factor 67.83 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.09375 1 0.09375 F (1, 30) = 5.561 P=0.0251 GPER1 Factor 0.2482 1 0.2482 F (1, 30) = 14.72 P=0.0006 E2 Treatment Factor 1.826 1 1.826 F (1, 30) = 108.3 P<0.0001 Residual 0.5057 30 0.01686

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.3617 0.1995 to 0.5239 Yes **** <0.0001

DMSO:gper1 +/+ vs. E2:gper1 +/+ 0.06643 -0.1076 to 0.2404 No ns 0.7287

DMSO:gper1 +/+ vs. E2:gper1 -/- 0.64 0.4725 to 0.8075 Yes **** <0.0001

DMSO:gper1 -/- vs. E2:gper1 +/+ -0.2952 -0.4732 to -0.1173 Yes *** 0.0005

DMSO:gper1 -/- vs. E2:gper1 -/- 0.2783 0.1068 to 0.4499 Yes *** 0.0007

E2:gper1 +/+ vs. E2:gper1 -/- 0.5736 0.3909 to 0.7563 Yes **** <0.0001

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Figure S6D: Male

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.223 0.3885 ns No ANAS Factor 10.18 0.0152 * Yes GPER1 Factor 3.311 0.1583 ns No ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.3103 1 0.3103 F (1, 54) = 0.7557 P=0.3885 ANAS Factor 2.584 1 2.584 F (1, 54) = 6.293 P=0.0152 GPER1 Factor 0.8402 1 0.8402 F (1, 54) = 2.046 P=0.1583 Residual 22.17 54 0.4106

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.3954 -0.2893 to 1.08 No ns 0.4266

DMSO:gper1 +/+ vs. ANAS:gper1 +/+ 0.5808 -0.1127 to 1.274 No ns 0.1308

DMSO:gper1 +/+ vs. ANAS:gper1 -/- 0.6772 0.0003279 to 1.354 Yes * 0.0498

DMSO:gper1 -/- vs. ANAS:gper1 +/+ 0.1854 -0.4251 to 0.7958 No ns 0.8518

DMSO:gper1 -/- vs. ANAS:gper1 -/- 0.2818 -0.3098 to 0.8735 No ns 0.5902

ANAS:gper1 +/+ vs. ANAS:gper1 -/- 0.09649 -0.5052 to 0.6982 No ns 0.9739

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Figure S6D:

Female

Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 13.15 0.0092 ** Yes ANAS Factor 5.314 0.0907 ns No GPER1 Factor 0.8928 0.4824 ns No ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 2.435 1 2.435 F (1, 46) = 7.39 P=0.0092 ANAS Factor 0.9838 1 0.9838 F (1, 46) = 2.986 P=0.0907 GPER1 Factor 0.1653 1 0.1653 F (1, 46) = 0.5016 P=0.4824 Residual 15.16 46 0.3295

Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value

DMSO:gper1+/+ vs. DMSO:gper1 -/- 0.5617 -0.04022 to 1.164 No ns 0.0753DMSO:gper1+/+ vs. ANAS:gper1+/+ 0.7289 0.07372 to 1.384 Yes * 0.0238

DMSO:gper1+/+ vs. ANAS:gper1 -/- 0.3993 -0.2026 to 1.001 No ns 0.3014

DMSO:gper1 -/- vs. ANAS:gper1+/+ 0.1671 -0.4664 to 0.8007 No ns 0.8953

DMSO:gper1 -/- vs. ANAS:gper1 -/- -0.1624 -0.7407 to 0.4159 No ns 0.8769

ANAS:gper1+/+ vs. ANAS:gper1 -/- -0.3295 -0.963 to 0.304 No ns 0.5141

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Table S1. PCR primers.

Gene Forward Primer (5'--- 3') Reverse Primer (5'- -- 3') LTR (5'--- 3')

gper1 TCAAGTTGCCGTCACAATGC GTCATCCTCTCCCTGTGGTT

ef1α GCGTCATCAAGAGCGTTGAG TTGGAACGGTGTGATTGAGG

mtor ATAAGAAAAGAAACCACATGTCATACC CTTACCACTCAGAGAGACCAAAG CCCTAAGTACTTGTACTTTCACTTG

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Table S2. RT-PCR primers.

Gene Forward Primer (5'--- 3') Reverse Primer (5' --- 3')

gper1 CTCGTGAATAAAGTGTTGCAG GCAGTCTTGTTTCCTCCAG

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Table S3. Morpholinos.

Gene Sequence (5'--- 3') Type Amount Injected (ng)

esr1 AGGAAGGTTCCTCCAGGGCTTCTCT ATG 2

esr2a ACATGGTGAAGGCGGATGAGTTCAG ATG 2

esr2b AGCTCATGCTGGAGAACACAAGAGA ATG 2

gper1 ACATTGGTAGTCTGCTCCTCCATGC ATG 2

gper1 GCTGCAACACCTGTTATAAGAGAAA Splice 2

mtor GGTTTGACACATTACCCTGAGCATG ATG 2

control CCTCTTACCTCAGTTACAATTTATA 2

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Table S4. Antibodies.

Antibody Application Concentration Supplier, Catalo g Number

α - PCNA IHC 1:200 Anaspec , AS-55421

α - Pan-Cadherin IF 1:1000 Sigma, C3678

α - BrdU Whole-mount IHC 1:500 Sigma, B2531

α - pEGFR (Tyr1173) WB 1:1000 Milipore, 05-483

α - Akt WB 1:1000 Cell Signaling, 9272

α - pAKT(Ser473) WB 1:1000

Cell Signaling, 4060 Whole-mount IHC 1:200

α - mTOR WB 1:1000 Cell Signaling, 2983

α - S6 WB 1:1000 Cell Signaling, 2217

α - pS6(Ser240/244) WB 1:1000

Cell Signaling, 2215 Whole-mount IHC 1:200

α - β-actin WB 1:5000 Cell Signaling, 4970

α-Rabbit (Alexa Fluor®647) IF 1:500 Abcam, ab150075

α-Rabbit IgG-HRP IHC,WB 1:1000 Santa Cruz, sc-2004

α-GPER1 IHC 1:50 Sigma, HPA027052

α -HNF4α IF 1:50 Abcam, ab55223

α- Mouse (Alexa Fluor® 488) IF 1:500 Jackson Immuno Labs

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Table S5. Chemicals.

Chemical Concentration Supplier, Catalog Number

β-Estradiol 10 µM Tocris, 2824

MPP dihydrochloride 80 µM Tocris, 1991

PHTPP 8 µM Tocris, 2662

G-15 60 µM (embryo)

Tocris, 3678 10 µM (adult)

G-1 8 µM Tocris, 3577

Anastrozole 10 µM Tocris, 3388

Fulvestrant, ICI 182,780 10 µM,15 µM (embryo) Tocris, 1047

10 µM (adult)

740 Y-P 2 µM Tocris, 1983

Rapamycin 1 µM Tocris, 1292

LY294002 HCl 15 µM Tocris, 1130

NSC 228155 5 µM Calbiochem, 530536

Erlotinib HCl 10 µM Selleckchem, S1023

MK-2206 2HCl 5 µM Selleckchem, S1078

Metronidazole 10 µM Sigma, M3761

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Table S6. Clinical Information for GPER1 scoring of non-cancer liver tissue.

Diagnosis GPER1 score to Reason for biopsy/resection (non-cirrhotic liver) E tiology of cirrhosis Age/sex

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sort

non-cirrhotic liver 1 Liver masses (final diagnosis of hepatocellular adenomas)

44F

non-cirrhotic liver 1 Metastatic colorectal cancer 43M non-cirrhotic liver 0.5 Metastatic melanoma 76M non-cirrhotic liver 0.5 Metastatic colorectal cancer 33M

non-cirrhotic liver 0.5 Liver mass (final diagnosis of focal nodular hyperplasia)

41F

non-cirrhotic liver 0.5 Metastatic colorectal cancer 73M

non-cirrhotic liver 0 Autosomal dominant polycystic kidney disease (wedge biopsy of liver taken during bilateral nephrectomy)

55M

non-cirrhotic liver 0 Liver masses (final diagnosis of hemangiomas) 36F non-cirrhotic liver 0 Liver mass (final diagnosis of hemangioma) 37F

non-cirrhotic liver 1 Liver masses (final diagnosis of hemangiomas) 52M

cirrhosis 0.5 Primary sclerosing cholangitis 27F

cirrhosis 1 Primary sclerosing

cholangitis and possible autoimmune hepatitis

24F

cirrhosis 2 Alcohol 39F cirrhosis 2 Alcohol 41F

cirrhosis 1.5 Alcohol 44M

cirrhosis 1 Non-alcoholic steatohepatitis

34M

cirrhosis 0.5 Primary biliary cirrhosis 45F

cirrhosis 0.5 Non-alcoholic steatohepatitis

52M

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Table S7. Clinical Information for GPER1 scoring of liver tumor and adjacent tissue.

Diagnosis GPER1 score to sort Paired Tissue Sample GPER1 score to sort Age/se

x Mixed HCC and Cholangioarcinoma 1 ANT (cirrhosis) 2 57F Mixed HCC and Cholangioarcinoma 1 ANT (cirrhosis) 1.5 58M

Adeocarcinoma 1 ANT (cirrhosis) 1 59M HCC 1 ANT (cirrhosis) 1.5 76M HCC 1.5 ANT (cirrhosis) 2 68M HCC 1 ANT (cirrhosis) 2.5 65M HCC 1.5 ANT (cirrhosis) 1.5 51M HCC 2 ANT (cirrhosis) 1.5 62M HCC 1 ANT (cirrhosis) 2 57F HCC 2 ANT (cirrhosis) 2 61M

Mixed Carcinoma of the liver 1 ANT (cirrhosis) 1.75 64M

HCC 1.75 ANT (cirrhosis) 1.5 73M

HCC 1.5 ANT (cirrhosis) 1.5 57M

HCC 1.75 ANT (cirrhosis) 1.25 59M

HCC 0.5 ANT (cirrhosis) 1 53M

HCC 0 ANT (cirrhosis) 1.5 66M

HCC 0.75 ANT (cirrhosis) 1 60M

HCC 1.25 ANT (cirrhosis) 1 49M

HCC 1.5 ANT (cirrhosis) 1.25 76M

HCC 1.5 ANT (cirrhosis) 1 42M

HCC 1.25 ANT (cirrhosis) 0.75 53M

HCC 0.75 ANT (cirrhosis) 1.25 72M

HCC 1.75 ANT (cirrhosis) 1.5 57M

HCC 2 ANT (cirrhosis) 1 69M

HCC 1.25 ANT (cirrhosis) 0.5 46M

HCC 0 ANT (cirrhosis) 1.25 59M

HCC 1.5 ANT (cirrhosis) 1 58M

HCC 0.75 ANT (cirrhosis) 1 40M

HCC 1.5 ANT (cirrhosis) 1.5 67M

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HCC 2 ANT (cirrhosis) 1 82M

HCC 1.5 ANT (cirrhosis) 1.25 71M

HCC 1.5 ANT (cirrhosis) 1.75 56M

HCC 1.5 ANT (cirrhosis) 1.5 57M

HCC 1.75 ANT (cirrhosis) 1.5 74M

HCC 1.25 ANT (cirrhosis) 1 46M

HCC 0.25 ANT (cirrhosis) 1.25 52M

HCC 1.5 ANT (cirrhosis) 1.5 49F

HCC 1.5 ANT (cirrhosis) 1 49M

HCC 1.5 ANT (cirrhosis) 1.25 41M

HCC 1 ANT (cirrhosis) 2.25 45M

HCC 1.75 ANT (cirrhosis) 1.5 65F

HCC 1.5 ANT (cirrhosis) 1.25 69M

HCC 1.5 ANT (cirrhosis) 1.25 45M

HCC 1.75 ANT (cirrhosis) 1.25 57M

HCC 1.5 ANT (cirrhosis) 1.75 62M

HCC 1.75 ANT (cirrhosis) 2 50M

HCC 1.5 ANT (cirrhosis) 1.5 67M

HCC 1.5 ANT (cirrhosis) 1.25 50M

HCC 1.5 ANT (cirrhosis) 2 30M

HCC 1.75 ANT (cirrhosis) 1.5 30M