www.sciencesignaling.org/cgi/content/full/2/72/ra25/DC1

Supplementary Materials for

Bruton’s Tyrosine Kinase Revealed as a Negative Regulator of Wnt–β-

Catenin Signaling

Richard G. James, Travis L. Biechele, William H. Conrad, Nathan D. Camp, Daniel M.

Fass, Michael B. Major, Karen Sommer, XianHua Yi, Brian S. Roberts, Michele A.

Cleary, William T. Arthur, Michael MacCoss, David J. Rawlings, Stephen J. Haggarty,

Randall T. Moon*

*To whom correspondence should be addressed. E-mail: rtmoon@u.washington.edu

Published 26 May 2009, Sci. Signal. 2, ra25 (2009)

DOI: 10.1126/scisignal.2000230

This PDF file includes:

Fig. S1. Bioinformatic overlap of independent small-molecule and siRNA screens

reveals previously unidentified regulators of Wnt–β-catenin signaling.

Fig. S2. Zebrafish expression and loss-of-function data.

Fig. S3. Protein interaction networks for BTK and CDC73.

Fig. S4. Context-dependent regulation of Wnt–β-catenin signaling by CDC73.

Table S1. Sequences of siRNA sense strands.

Table S2. Descriptions of supplementary database files.

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencesignaling.org/cgi/content/full/2/72/ra25/DC1)

Databases S1 to S12, S16 to S18 (Microsoft Excel format)

Databases S13 to S15 (cys format)

ERCC1GTF2H3XPA

CDK7

COPS2RPA70

RPA3XPC

EWSR1

RAD9B

RFC3

RFC4

DNA2L

RAD17

HUS1

RFC2

POLD3POLD1

CDC5L

GEMIN7

ADCYAP1R1

PDE10A

CLK1

CD3Z

GYS1

PPIG

CALML6

CNGA4

EPAS1DDX52

NPPB MYL2HMOX2

GTF2E2MNAT1DDB2

GTF2H4

CCNH

GTF2H2GTF2H1

UBL5

CDT1

MCM3CDC7

ORC2LMCM10

ORC1LMCM4

MCM5MCM7

BCL2L11PRKG1

CNGA3

BLVRB

PLRG1 NAT1

BLVRAPOLD4

APEX1

RFC5

POLD2

MEF2CENSP00000265728ORC4LBM28

CDC6MCM6

ORC6L

ORC5LORC3L

CAMK2A

TUBB

ENSP00000291295

KIAA0828

CNGB1

CAMK2BCAMK2G

13-ketooctadecadienoic acid

CHRM1

HRH1

pachyrrhizin

HNMPA-(AM)3

DYRK4

WNK4

TACC3

CGI-61

GPAM

ESCO1

PRKG2

MDM2 Inhibitor

methoxyvone

carbinoxamine

obliquin

daphnetin

phenazopyridine

MDM2

SGPP1

KCNQ3

PPAP2ARFXANK

STARD10

HACE1

SNTB2

UROS

UGCG

EMP3

ATP6AP1

DAGLB

CXCL8

SU-4312

alpha-humulene

Rho-Kinase Inhibitor

NOS2

eupatoriochromene

DCTN1

MAP3K11

cytochalasin J

stearoylcarnitine

PDE2

PRKD

EHNA

PDE1

2-methoxyxanthone

"2,3-dihydroxy-6,7-dichloroquinoxaline"

8-methoxymethyl-IBMX

"7,4'-dimethoxyisoflavone"

helenine

PSMD14

SGPP2

ENSP00000353887

PSMD6

ANKRA2

CDH26

MAT2B

GATA1ENSP00000242108 FAS1

FRKCSTA

CDKN3

BLM

ADCY5ZNF622RBBP4

ZBTB16

CCNB2

RBBP8

RAB3IP

SAP30

RPH3ALSIN3A MTA3

RBBP7

ENSP00000230731

MBD3MTA2

MDC1

NALP2H2AFX

MRE11A

ATMENSP00000343741

XRCC5

ENSP00000272317

SND1

MYBL2

G22P1

MLH1

CDC25AMSH6

GNAS

CHEK2GNG5

MSH3

CHEK1

PTPDC1SLC26A10

RBL2

GNG7

FKBP3

TSSK1

DNMBP

GCGR

PRKXAHCYL1KCNV1

SPAG5

ENSP00000321160

RAB3D

SUV39H1

ENSP00000248054

NCOR2

MTA1

NOTCH4

GRB2

RUVBL1

SYK

MAPK9

CRKRAD23ACRKL

ERBB2

FEN1

POLE

DNAJC7ENSP00000231383

ENSP00000263274

RAD9A

NDUFB2

NDUFA8NDUFB7

NDUFC1

RFC140

ENSP00000252576NDUFB5

GALT

NDUFB3

LALBA

NDUFAB1NDUFA2

NDUFB4NDUFA1

ENSP00000319149

PSMA6 PSMB2

PSMA5

PSMA7

PSMB4

PSMB7

PSMB8

PSMA3

PSMB1PSMB9 PSMB3

PSMB6PSMA1 PSMA2MAT1A

MMP14

RECK

XRCC2

TSNAX

GATA2

TIMP2

STARD6

RAD51L3

MMRN1

XRCC3

KIF22

TOPBP1

B4GALT1

RDH13

HSD3B7

RDH11

GALK2HSD17B12

GALERDH12

TP53BP1

RAD50

CCNE2 CDC34CCNA1

UXTCCND3ERCC3

POLR2I

NR2F1 RAD23BCALML3

POLR2B

ERCC2

BARD1

MEF2A

SH3BP5GEMIN5

DDX20NR2F2 WT1KIF1C MSH2

GEMIN6

GEMIN4

ENSP00000300413

SNRPD2

SNRPBPRPF4

GEMIN2

PAK2

RIPK2RPSA

PTPN21

RPS18

TNFRSF1AINT6

B3GAT3

BAK1

BMX

BIRC2

RPL18CD40

RPL6

MPP3

CHGN

AKAP1 BAXBBC3

GNG2RIMS1

RIMS2GNG4MSH4

EXO1

GNG3

GNG13

SNX13

KIF2B

EXTL3

SPAG9

B3GAT1

B3GAT2

SMURF1

CHGN2

BID

CFLAR

EXT1

CARD4

EXT2

CASP1

BAG4

CASP8

FADDMAP3K5

TANK

GNG10

RALBP1

ADCY8CRADDGNB3

MSH5

GNB1

RPS20 RAPGEF4TREX1

RPS21

RASSF5

EIF3S7

EIF3S2

TNFAIP3RPS5

GNAI2

RIPK1GNAI1GNAI3

TRAF1

TRADD

CASP2

ENSP00000268605ETNK2

GNGT1

CDK4

PCNA

CDK6

CCND1

CCNA2

CDKN2A

PTK2

CCNB1

CDC2

CDKN1A

CDK2

PTPN6

PDE6B

NPPA

ENSP00000354532

NPR1

NTRK1

ADCY2ADCY1

GNRH1ENSP00000308541

RB1

IRS1

PDE1B

PDE3B CCND2

JUN

SPP1

IL4

JAK3

KDR

VEGF

LOX

FUCA1MGAM

SRF

MYLK

LATTEC

ACTG1

BTKSFRS6

ADCYAP1AHCY

MAT2A

CTSL GSK3B

ADCY4

SHC1TGFB1

GSK3ACOMT

PDE7B

PRKDC

HDAC2 PRSS3

LCK

PLG

INSR

IRS2

RORA

CASP3

ENSP00000276449

RAD51L1

RAD51C

H1F0

IKBKB

HDAC5

PLOD3

VDR

HDAC11

CFTR

BIRC3

IL2

ESR1

SOCS1

TFRC

CD4

MMP2

CSF2

FOS

SOCS3

FYN

EGR1

RXRA

NR1I2

HDAC6

RXRB

CREB1

BCL2

GJA1

MAPK8

ROCK1

KIAA0511

ACO1

RAF1

TP53

EGFR

BIK

BCL2L1

HMOX1

RAD51

HDAC3

LYN

CYP11A1

HDAC1

VCL

PTPN1

SRC

SMARCA4

PIK3R1

DNMT1

bisindolylmaleimide II

CTRLthioflavine S acriflaviniumlactacystin Exo1

suberoylanilide hydroxamic acid

AAF

APRT

uracil

hypoxanthine

thymine

pyrophosphate

phosphoribosylpyrophosphate

guanine

adenine

SLC40A1ENSP00000341714 terbutryn

epoxomicintauroursodeoxycholic acid

chrysamine G

ENSP00000351801

3-nitropropionic acid

FTL

chromomycin

actinomycin D

BVD

hydroxyurea

mitomycin C

acyclovir

rimantadine

5-iodo-2'-deoxyuridine

trifluorothymidine

bromodeoxyuridine

guanosine

cytosine arabinoside

NT5C2

uridine

ribose 1-phosphate

thymidine

cytosine

inosine

bisindolylmaleimide I

ADA

"8-beta-hydroxycarapin-3,8-hemiacetal"

aminothiazole

CD248ononetin

carbenicillin

Ucf-101

ACTA1

bistheonellide A

"epoxy (4,5alpha)-4,5-dihydrosantonin"

ptaeroxylin

JAK3 Inhibitor VIcytochalasin R

5'-deoxy-5'-methylthioadenosine

FTLL1 TFR2

deferoxamineHIF1AIREB2

HTRA2

"leukotoxin B (12,13-EODE)"

PPP3CA

6-mercaptopurinetyrphostin-8

RPS6KA3KCNN3

dequalinium

cetylpyridinium chloride

clotrimazole

DRD5

SST

CRH

NPY

GANAB

CD22

"3,7-dihydroxyflavone"

4'-methoxyflavone

RWJ-60475-(AM)3

PTPRC

CD2

ethanol

tetrahydroisoquinoline

TH

salsolinol

TIQ

1-carboxysalsolinol

acetaldehyde

tetrahydropapaveroline

ENSP00000302150

"quercetin tetramethyl (5,7,3',4') ether"

anorexigenic peptide

1-(2-hydroxy-4-morpholin-4-yl-phenyl)ethanone

xanthyletin

butenolide

picrotoxinin

picrotoxin

CCK

ST13

3-hydroxybenzylhydrazine5-hydroxyindoleacetic acid

alpha-methyl-p-tyrosine

levodopa

SLC17A5

dopamine

HTR1A

5-hydroxytryptophan

SU9516

DDC

apomorphine

N-n-propylnorapomorphine

fenoldopam

3-PPPpiribedil

DRD2

ADTN

pergolidequinpirole

CYP2C8 CYP26A1all-trans-retinoic acid

13-cis-retinoic acid

FG 7142

GABRQ

RARG

pentylenetetrazol

RETINAL

"eicosatrienoic acid(20:3,n-3)"

ZK 93426

bicuculline

RARA

TFPI2

PRDM2

5-aza-2'-deoxycytidine

RARB

AM-5809-cis-retinoic acid

RXRG

retinoic acidTTNBP

RASSF1

DAPK2

TPSD1

ACE

RHC-80267

lomofungin

MPN

DAGLA

PTPRCAP

SEMA4D

LLH

hydralazine

PENT

PLOD2

ENSP00000272190

PPRC1

KITLG

TL1PDCD5

EGCG

FUCA2IL5

theaflavin monogallate

AK5

MMP9

crassin

IB-MECA

ADORA1

BIRC1ADORA2A

ADORA2BADORA3

terreic acid

adenosine

PLN

quinacrineCTSB

PRNPSERPINA8

leupeptin

NUP214

TRH

ENSP00000275292

malonyl-CoACO2

coenzyme A

"2,3-diacetoxy-7,8-epoxy-24,29-dinor-1,3,5-friedelatriene-20-carboxylic acid"

TPSB2

stilbene

pinosylvincinnamoyl-CoA

CHS

CD44

PTGER3

GCG

prostaglandin

11-deoxy-PGE1

PTGER1

PTGER4

"16,16-dimethyl-prostaglandin E2"

PGE2

HDAC9BML-210

HDAC10depudecin

HDAC7

HDAC4

HDAC8trichostatin A

MEF2D

naproxen

ketoprofenpiroxicam

meloxicam PTGS1

indomethacin

CYP2C9

ibuprofen

tenoxicamdiclofenac

IL8

PHIP

CNR1

NFKBIA

2-arachidonoylglycerol

TNFENSP00000236138

"anandamide(18:2,n-6)"

TRPV1

PTGS2

IL6

chalcone

cafestol

diterpene

capsaicin

ikarugamycin

kahweol

CAPNS1

trichodion

IL1Acurcumin

IL2RA

PDE

Ro 20-1724cyclic AMP

forskolin

PRKAR1A

PRKAR2B

IBMX

rolipram

"5-[(DIMETHYLAMINO)METHYL]-3-(1-METHYL-1H-INDOL-3-YL)-1,2,4-OXADIAZOLE"

PDE4DPDE3A

cytochalasin DINS

POMC

SLC2A4

jasplakinolide

THY1

PTHLH

latrunculin B

GRB10

CYP24A1

vitamin D3

"24,25-dihydroxyvitamin D3"

calcitriol25-hydroxyvitamin DCYP27B1

CALCAPMF1

calcium

kawain

vinblastine

CYP3A4

ABCB1

ENSP00000253475

ENSP00000273951

PTH

vitamin D

thiazide

topotecan lactone

camptothecin

hydroxycamptothecin

"10,11-methylenedioxycamptothecin"

NSC610457

9-aminocamptothecin

7-chlorocamptothecin

NSC610459

aloisine Aroscovitine

NPY6R

GLB1CDK1

kenpaullone

CDK5

MAPKAPK5

NOS2A

naringenin

GSK-3 Inhibitor II

SLC5A5

galangin

pinocembrin

pinobanksin IFNG

luteolin-7-O-beta-D-glucuronideluteolin-7-O-glucoside

eriodictyol

luteolinindirubin

CYCSchrysin

chrysoeriol apigeninflavoneEDN1

UGT1A5 AHRindigoacacetin

CYP1A1PPARG

S-adenosylhomocysteine

quercetin

linarin

CYP1B1

TOP1camptothecin derivativeTrkA Inhibitor

bisindolylmaleimide III

aaptamine

cuneatin methyl ether

NSC610456

bisindolylmaleimide IV

genistinuridine diphosphate

GAMTbiochanin A 7-O-beta-D-glucoside

creatinine

creatineN-methylhydantoin

ammonia

ABP1

SCNN1B

amiloride

Na+

phenamil

benzamil

EIPA

methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate

4'-hydroxyflavanone

TMB-4

pralidoxime

Hloe 7

HI-6

obidoxime"2,3-butanedione-2-monoxime"

MYLK2ENSP00000304701

CYB5

H2O

biochanin A

NADPdihydrobiochanin A

cyanidin

leucocyanidin

NADPH

ENSP00000265171

HBEGF

TDGF1RG-13022

ELTD1

EREG

TGFA

ARFRP1

"L-744,832"

Chromozym TH

chromogenic substrate S-2288S-2266

FDPS

8-methylgenistein

S-adenosylmethionine

epicatechin

genistein

1H

PDE2A

STMN1taxol

colchicine

TUBB1

PDE2B

HTR1D

ketanserin

sumatriptan

CP 93129

GR 55562

HTR7

HTR1F

HTR1B

HTR2A

SKF 38393

GRIN2A

GW-5074

DRD1

NSD 1015

N-0434

scriptaid

MGMT

SULF1

DCTN2

KIAA0494

TRPV6CACNA1G

NR1D1

PMS2L4

TRPC1

CACNA1E

CDC2L5

MYLK4

ENSP00000233615CACNA1I

FALZ

LSP1

VPS4A

TP73

YAP1

CACNA1B

MAZ

NCOR1

YES1

SHAX3

NFE2

TP73L SHAX1

PREPL

ENSP00000255108

TIMELESS

SLC7A6PTPN22

ENSP00000312402

ENSP00000295488

MGST2

STK38

MUSK

GSTM5CRK7 HECW1

PLA1A

NME1

FOLR1STK39

SHBG

APEH

SLC12A2STK32A

ENSP00000330408

ASCC3

RUNX1

UBE1DC1

ENSP00000257215CPM

SAE2

PCIF1

BNIP3

RPL11

RHOC

DOLPP1

ENSP00000282947

GNA13

RPN2

DCC

ITGB5

TCP1CCT5CCT7

ENSP00000306100

SLC30A4

PMS2

CCRKUBE2T

DCAMKL1

KCNQ4

DCAMKL2

ARF3 CD47

ITGAVCCT3

THBS1

VTN

CYR61

ITGB8

ABI1

LRRK2RAP2B

CDCA2

RHAG

CLCNKA

PLCE1ARFIP2

NVLPPM1M

PDE6DKCNA10

PTPN23

TTBK1

SUV39H2KIAA0256

TTK

FBXL12SGOL2

NEK4ECE1

ARFIP1

ITGB6

ENSP00000302882RAPGEF3

KBTBD7

SUA1

P2RY2

NOVHPTPNS1UBE2I

ITGA2

DIAPH3

BLMHITGB1

CCT4ITGB3

CCT2

CCT8

ITGA6

CD63

GGA2

APBB3

CCT6A

USP6NL

PSCD3

ENSP00000229239APOEBACE2 NGEF

APBB1

EPS8TLK2

APPBP1LRP1

ACHESERPINA3

APBA3

ENSP00000349960

EPHA4

ACTC

EFNA3APBA2

APBB2

BCAT2

DDX54

ZW10

ZWINTHUWE1

TRPV2

BRD4ARK5

ROS1

ENSP00000290786

GRK7

LPHN1

ENSP00000306328

ENSP00000011684EFNB3

PLSCR1VGCNL1

TRPV3

UBE3A

UBE2G2ENSP00000342589

AMFR

EFNA2

ENSP00000340474

ARHGEF15

UBE2G1

EFNA1

ENSP00000350232

CDK2AP1

ENSP00000301488

MYD88

ANGEL1

CBX5

BXDC1P

CDKN2C

CDKN2B

ENSP00000264864

TRPV5

DNCL1

UBR1

PKIG

TNFRSF13BADCK2

STARD8

KCNC4

EFNA4

BCAT1

DAZAP2

PARK2

BCKDHB

BCKDHA

CD9

CD6

ALPK2

CD81

ALCAM

ICAM1

VCAM1SLC9A1

SLC9A3R1

PBX2

ICAM3EFNB2

ARCN1

IRAK3MAP3K14

COPA

IRAK4IRAK1

COPB2

THOC2

PAPSS2COPZ2

COPG

ENSP00000313643

SUOX

COPB

TNFSF13B

MLL4

ATP1B3

STX5A

RCV1 ENSP00000352872MKI67

MKI67IPSTARD13

GRK4UFD1L

MAPK8IP1RASA1

DUSP10

PAK7

EFNB1

ABL1PRKCABP

ZNF567EFNA5

APP

APBA1BACE1

DOCK1PRKCQ

VANGL2 ENSP00000267569PLD1

ELMO1

FRAT2ARHGDIA

VAV2

AKAP13

SH3BP1RHOA

SRGAP1

OPHN1

GNG12

ARHGEF6

ATN1

RAC2

PLXNB1ARHGEF7

JUNDBAIAP2

RTKN

RHOG

RHOQRHOBTB2

GNA12

POLIPLXNB2

ARHGEF9ARHGEF16

WASF2

WASF1

ARHGAP1

DIAPH1

CDC42

PTDSR

ARHGAP5

PKN1

ARHGEF2

PTP4A1

ARHGDIB

CDC42EP2

ATF3

NCF2FOSL2

BCL10

PLEKHG2

RHOV

ARHGEF8

MALT1ENSP00000304283

ARHGEF4

ARHGEF12

VAV3

DDOST

GCS1

RFWD2

JUNB

RPN1

FOSB

PSMD4

PKDREJ

ZP3

GAA

PSMB5

SLC12A3

ENSP00000260662

HMBS

GBA3

AGL

PRO1400

PSMC5

STARD5

PSMD12PSMD7

TCOF1

WKL1

MAPT

MYL3ATP5F1NRK

ATP5J2ENSP00000318086

TMPOATP5H

CDKN2D

DDX17

PTPRGENSP00000352232

ATP5J G9A

FZD4

FZD8WNT3A

FZD7FZD6

FZD9

FZD1SUCLG1

SUCLA2

MDH2

LOXL2 ADC

RARS

WNT4TPI

ACY1

ARG2

ENSP00000234111

MTHFD1

ARG1

ENSP00000348342

CD5

FREQ

PIK3R4

SOCS5

TAT

TLR9BECN1

ALDH1B1

ENSP00000218516

YARS

SRPK1

ENSP00000303246

ASS

MMIF

ACY3

ACLY

BBOX1

CPSF1 SFRS5

CPSF2

PABPN1NFX1

CSTF3

GALK1

PHF5ACSTF2POLR2E YB1

PRPF8

SF3A2PAPOLA

SF3A1

SNRPD3

NHP2L1

CD2BP2 WDR57FUS

DLST

FZD2FZD5

SFRP4

ALDOBALDOC

ENO2

PKM2

SFRP1

SFRP2

MUT

MDH1

ENSP00000334713GATM

PSMD2

PSMC3PSMD5PSMD3

PSMC6PSMC2

PSMC4PSMD13

ENSP00000264382LCT

PSMD9PSMD1PSMC1

PSMD11AMPD1

AMPD2

GLO1

GALC

ENSP00000350063

CERK

SMPD3DEGS1

UGT8

ASAH1

DEGS2

PPAP2B

SMPD2

SORD

IDH3AIDH3G

IDH3B

IDH1ALDH7A1

ALDH9A1

ADSS

IDH2

GOT2

ENSP00000257347

ENSP00000239123

ACO2

EPRS

GLUD2NAGS

CDO1

ENSP00000235407

GCLM

GCLC

GAD2

ALAD

PTPRN

ALAS1

FECH

UROD

CPOX

ALAS2

KCNQ2

ASMT

HIBADH

GLUD1

CLYBL

ALDH18A1

ALDH6A1

ALDH2

DCXRAMPD3HPD

GBA

ATIC

ADSL

AKR1B1

PGAM2CARKL

PORCNCDA

ADPN

GPD2

GSTM2

GSTM3

MTHFD2

MTHFD1L

ENTPD1

GUK1

PKLR

NME3MCEE

NME4

ITPA

DUTENSP00000341279

UPP2NT5CDCTD

AICDA GNPATTK1

NT5E ITGB1BP3CANT1OAT

RRM1RRM2B

RRM2

GUCY1B2PNPT1

DHRS7

ENSP00000309735

ENTPD2

DHRS2

BFIT

DHRS1

DHRS3

GUCY1A2

NUDT2GCH1

TYMS

ENTPD3NT5C1A

ESCO2 MYST3

MYST4BPGM

NT5C3TE2

DBTOTC

UPB1

ABAT

CSUCK

NIN

CASP6

EMDVEGFC

FLT4

HRK

SNRPE

SFRS11SNRP70

U2AF1

LSM2

SF3B1

U2AF2

HNRPK

SFRS2

SFRS1

LMNB1

ATP5A1ATP5D

ATP5C1ATP5I

NXF1

SRPK2

DDX23

ASCC3L1

EFTUD2

ATP5G1LMNA

ATP5EATP5L

ATP5O

ATP5B

ATP6

FBXL10

CLOCK

FKBP1AS100A10

CACNA1A

MR1

ARNTL

KIF17

GUCA1ABST1

CDA04CREM

BC2

VPS4BRYR3 SLC25A18

FHL5

CACNA1H

NRIP2

GTF2I DINB1

KIF3CWNK1

FAM61B

ENSP00000281080

HCN1

RHOD

POLL

FARP2

RHOH

RHOB

MEN1

ENSP00000296214

MORF4L2

REV1L

HTATIP

MRGBP

MORF4L1

ING3

LCP2DMAP1

SH3BP2

RUVBL2

GAB2CBL

NOTCH3

BCAR1

FRS2

PLCG1VAV1

IGKCENSP00000290575

C1S

GBE1C1QA

LYZL2

C20orf27

IGKV4-1

E2F2

C2

SCAND1

AMY2A

HM13

SLCO4A1

IGLC1

CIDEA

ENSP00000248008

BRD2

SPG4ENSP00000272223CD74

CANX

DHX35

PEX3

ENSP00000272037

KCNH3

PEX13

PEX11B

GAS3

PLAA

PEX19

PEX12

NR6A1

MPZ

GOLGA4

PEX11A

TCRAPEX14

CST7

ARL1

PGM1

NCBP1

PPP1CAFMR1

PYGB

STAU

PHKG1CGI-69

PHKB

PHKA1

PHKG2

KCNC3

PHKA2PYGM

EMP2

LRP3ENSP00000292296

KIF3A

RAB6IP1MEST KIF3B

ADRBK1

RORB

KIFAP3

HCN2

C1QB

RENT1

GYS2

ABCD2PYGL

C4B

PGM3

C1QG

CASC3

CD38

ABCD1

ABCD3PEX16

ENPP2

SERPING1

NUP37

AAAS

ITPR3

ITPKA

POM121

NUP153

RAE1ITPR1

DDX3X

XPO1

SLC9A3R2PPP1R1A

TAF12

SUPT3H

TAF2

MYL6

PPS

TARBP2

NPM1

ILF3PRKRA

ROCK2

DNAJC3

ITPKB

INPP1XPV

MINPP1

MAP4K4

IPMK

CALM1

PIK3R3

BLNK

FCER1G

ENSP00000330620

TAF9

TAF6

TAF7

TAF1

TAF10

TAF5TAF4

TAF5L

CSE1L

IGF1R

F7

CDIPT

GGCX

PPP1CC

F10

F9

NGFR

ENSP00000233668

ILKHCST

PIK3CA

CD33

NR3C1

JAK1

IFNAR2

PMC2

TYK2

JAK2

IFNA2

IFNA1

IL2RG

GNA11

GNAQ

CSF3RSTAT5A

PROC

STAT2

PIK3CB

STAT1

HRAS

NGFB

PTEN

PLCB3

FURIN

PROS1

GRAP2

PDGFRB

TRRAP

CD19

YEATS4

ACTL6A

EIF2AK2

NOTCH2 SH2D2A

ILF2

WAS

AKR1A1

SOS1 GAB1

INPP5D

PIK3R2

KIT

PLCG2

PIK3CG

PIK3R5

PTPN11

NEDD9

RAPGEF1

CDCA3

NCOA6

RNPC2

CACYBP

FBXO4

FBW8

SKP2

ESR2

CDKN1B

CUL1

CDKN1C

STAT3

IFNAR1

NR0B2MAD2L2

BUB1

PIAS1

NEK2

APC10UBE2D1RBX1

CDC16

BUB1B

ENSP00000297808

GNRHR

BUB3

APC5CDC27

UBE2C

ANAPC4

FZR

CDC23

ANAPC7

ANAPC1

ANAPC2

ENSP00000344538

AP4B1MAD1L1IFNW1

AP3B1MAD2L1CDC20

SLC25A23

HCK

SERPINB2

HLA-J

KIF1A

MAGED1HLA-F

ENSP00000238018

TOMM70ACDC91L1

PLAURPIGO

ENSP00000232003

PRPS1

ENSP00000346809PIGF

GPAA1

CD8AGRP58

PIGT

PIGS

TAP1TAPBP

PIGKBCL3

ENSP00000311750

TUBA1Y1

B2M

KIR3DL2

ENSP00000297933

ILT4HLA-A

TRBV21-1

ENSP00000299064

HLA-B

G6PC

PPP1CB

PRPS2

ENSP00000223366 SNF1LK2

ENSP00000305873PLAU

TUBB6

SDHA

ENSP00000261755SERPINE1 TTC1

PSIP1

NUP98

NUP107

HMGA1

NUP160NUP133

ENSP00000296289ROR2

PMI1

TKTL1ENSP00000280605

SDHB

TPR

BANF1ENSP00000341934

PCNT1

NUP88

ARA24

NUP155

KPNB1

PFKLENSP00000327074

ENSP00000290573PFKM

ENSP00000292432

PBEF1ENSP00000354710

WNT5AFH

PLIN

PPP1R9B

ANTXR1

ANTXR2

FZD10

LRP6WNT1

ENSP00000343752

KREMEN2DKK4

ENSP00000240093

DKK2

DKK1

GNRH2 VHL

MCC

CUL5

KIF23

CUL2

PLK1

POLR2G POLR2FPOLR2L

GTF2B

POLR2CSUPT5H

POLR2JPOLR1C ENSP00000225507

POLR2HPOLR2KCSTF1

CCNT1

CTDP1

POLR1D CPSF3

POLR2D

CDK9POLR2A

APRIN

CASP7

TCEB3

ASB8

SIR2L

ITM2C

ENSP00000303427

RAD21SA1

TCEB1STAG2

TCEB2

REC8L1

CDC25C

IFNB1

VDU1CSPG6

RNF7

SMC1L1

PPP1R8

IL13RA1

IL4R

IL13CSF3

CUL4A

PIK4CA

ENSP00000277541

PI4KB

ENSP00000322455

PROZ

SYNJ1OCRLSYNJ2

PIK3C3

GAS6

KPNA6

E2F5

YWHAE

NRAS

RAP1B

RRAS

TERT

MYC

E2F1

PRKCB1

TFDP1

KRAS

RBL1

SP1

YWHAQRAP1GA1

DOM3ZENSP00000337354

SKIV2L

LPL

SEN15

EXOSC4CEL

MGLL

AP1S1

JPH4SC5DL

EIF5

PDRG RRAS2MAP3K2

MAP2K5

MAP3K3

TLR3CHUK

BAD

PRKCA

RAP1A

FASNFOXH1 TGFB3TRAF7ACVRL1 YWHAB

AP1G1

MCL1BCL2L2

PPP3CC

ENSP00000278568SERPINB3

IKBKG

TRAF6

AP1M2BCL2A1 ENSP00000265433

CCNE1

HMG20BBRCA1

SHFM1

EMSY

WEE1

FANCGBCCIP

FLNA

FANCD2

THRBGSBS

PPP2R1A

IGBP1

PCAFPRKCI

FBXO7

BRCA2

ACOX2

ENSP00000296088EXOSC6GTF2IRD1

ENSP00000252622STK11

ACSL4ACOX1

LSM1 ACVR1ENSP00000241416

EXOSC2MPZL1ENSP00000253458

SKIV2L2

EXOSC9

PLAGL2

CYP7A1

EXOSC8

DHCR7

DNAJC14SOAT1

TGFBR2TGFB2

SKI

YWHAZSKIL

MARK2SMAD2

MRAS

ACVR1C

ZFYVE9

SMAD6

CEBPB

TGFBR1

PFDN2

EXOSC3EXOSC10

EXOSC1

EXOSC7MAP3K7IP1SMAD3MAP3K7IP2

PPP2R5C

YWHAG

MAP3K7SMAD4

YWHAH

PPP2CBTSC2

PPP2R2A

AKT1

PML

EIF4E2

AKT3BRAF

SMURF2

EIF3S9

ACVR1B

SMAD7

CDC37

DAXX

SMAD9

BSP1SMAD5

RPS6KB1

AKT2

PIN1

MAP2K6PDPK1

MAP2K3

EIF4EBP1PKACA

RHEB

CCNG2

RPS6PPP2R5B

TSC1NEFL

EIF2S3

EIF2B1

EIF2B4

MLLT4

CREBBP

EIF4E

RPS6KA1

EP300

MAPK1

FRAP1

EIF4B

EIF2S1

EEF2K

EIF2S2RPS6KA4

MSK1PDK2

PPP2CA

ENSP00000301724

CARM1

DHTR

NCOA3MAPK12

MKNK1HD

CTBP1MAPRE1SKP1A

SIAH1

FBXW11MAPK11

NCOA1

ELK4

MKNK2

BTRC

CENPA

RPIA

DDX25

BDH

H6PD

SNX6

ASF1AKIF18A

TLK1

PECI

HMGCL

ME1

PDHXDCI

EHHADH

PGD

AGXT2

ENSP00000265838

PGLS

HADHA

HIPK2

PRPF4BENSP00000336655

ACADL

THOC4

IGHMBP2 CPT2BMP7

CAB39

ACADM

F11RPVRL4

INHBCACVR2B

INHBB

PVRL1

DLDOXCT1HMGCS1

ACADSACTL

PDHB

PDHA2

PVRL2

PVRL3RNF11

PDHA1

SORBS1

DLAT

BMP2

ATP6V1G2

LSM8

DDX39

CPT1A

ENSP00000300291

SOAT2 INHBA

HMGCS2HSD17B4

IRS4NRBP1

HADHSC

MAXHADH2

NFATC2PPFIA2SHC2

ACSS2

EIF2B5EIF2B2

EIF2B3

SSX2IP

HSPCA

CLDN7

VCP

FGF1

VIL2

RDX

MSN

FAF1

TJP2

MAOA

MEIS1KCNAB1

SNF1LKVAPA

MYT1ENSP00000259722

PTPRS

MEIS3 KCNA4

MEIS2

HOXA13

GPR125CBX3PSD95

ENSP00000334176ENSP00000280241

KCNA2

LSM6

SAV1

LATS1CHD2

MCRS1

MLL

HCFC1

PRPF31

RAB38

ADNP

KIF21BZNF265

PAPSS1

DHX8

HFE

ACADVL

RPL5

ACTN4

ACTN1

UBE4B

YAF2

PBK

MXD1

ZNHIT1

ACSS1

EDNRA

KIAA1232

NSFL1C

KIF1BMST2

GRK6

TEX10ASH2L

SENP3

ENSP00000293804

ENSP00000262419

RBBP5C18orf37

PHF20ENSP00000350303

PPFIA1

FRAT1

MET

CSNK1E

ENSP00000247161

TCF7L2

ATF2

TCF7L1 AXIN1

APC

ENSP00000314228

MAP4K1

SHC3

DUSP16MAPK8IP2

MAP3K10

VANGL1

DAAM1

MAPK10

RAC1

CXXC4

RHOF

CNNM4

CARD11

PXN

RHOU

LFNG

DVL2

CDHP

MAP4K2

CTNNB1

GLI

MAP2K1

LEF1

CSNK1A1LPTPRR

ENSP00000233607CENTA1

TCF7MAPK3

DVL1

AXIN2

ZAP70

MAP2K1IP1

SUFUPPP2R5D UBA52

DVL3

IQGAP1 CSNK1A1

MAPK14

TBL1X

NMI

JUP

MAP2K2

ARRB2

MAPK8IP3

MAP2K4

MAP3K1

ENSP00000340257

ZIC2ALDH3B1

MTSS1

PSEN1

EPHB2

CSNK PTCH

PTPRFOCLN

MYCN

CDH2CTNNA1

MAOB

CSNK2A2CA9

UVOCK2A1PTPRBCSNK2B

PGEA1GRIA2

PTPRM

CTNNA3FER

CDH5

CTNNBIP1SEF2

TJP1RNF111

ACP1

PTPRK

ARAF

PECAM1

Bioinformatic Overlap

Up in siRNA screen

Down in siRNA screen

Interactor not present in siRNA screen

Interactor not hit in siRNA screen

BTK

Reported interactions of siRNA screen hits Reported interactions of small molecule screen hits

Up in small molecule screen

Chemical interactor not present in small molecule screen

TEC

Supplemental Figure 1. Bioinformatic overlap of independent small-molecule and siRNA screens reveals previo sly of Wnt/β-catening signaling.unidentified regulators

u

Supplemental Figure 1. Bioinformatic overlap of independent small-molecule and siRNA screens reveals prev- β-catenin signaling. Integration of the protein interaction network generated

direct targets of siRNAs considered hits in the siRNA screen. Proteins in the network are represented by

pathway or small molecules that inhibited the pathway. Green nodes represent proteins the pos-pathway: siRNA targets that when silenced signaling was decreased. Grey nodes represent

molecules that were not present in the small-molecule screen.

iously unidentified regulators of Wnt/through a STRING analysis of targets of the siRNA screen hits totaling 1855 nodes (Left) and the small molecule and protein interaction network generated through a STITCH analysis of the small-molecule screen hits totaling 292 nodes (Right). Proteins common in each network represent the bioinformatic overlap totaling 108 nodes (Center). Of the overlap nodes, 34 were rounded square nodes and small molecules are represented by diamond nodes. Edges represent an interaction bet-ween two nodes. Red nodes represent negative regulators of the pathway inhibitors: siRNA targets that when

proteins not considered positive in the siRNA screen, and white nodes represent proteins that were not targeted in the siRNA screen or small

silenced activated the itively contribute to the

Supplemental Figure 2. Zebrafish expression and loss of function data.

48 hpf21 hpf14 hpf70% epiboly

0.0

0.4

0.8

1.2

1.6

Shi

eld

14 h

pf

21 h

pf

52 h

pf

Qua

ntifi

catio

nbt

k R

NA

vers

us a

ctin

btkactin

Ladd

erS

hiel

d14

hpf

21 h

pf52

hpf

-cD

NA

Ladd

er

btk

sensesense sense sense

btk btk btk

Cont

rol M

O

wnt

+ b

tk M

O 2

(1ng

)

wnt

+ C

ontr

ol M

O

wnt

+ b

tk M

O 2

4ng

)

Per

cent

Phe

noty

pe: z

ebra

fish

0

20

40

60

80

100n=75 n=56 n=67 n=52

Morpholino

PCR Primer

MO2 MO1

Amplicon AAmplicon B

E2 E6E5E4E3

Con

trol M

O

btk

MO

1

Amplicon A

Amplicon B

actin

Con

trol M

O

btk

MO

1

btk

MO

2

A

B

ED

C

Supplemental Figure 2. Zebrafish mRNA expression and loss of function data. (A-C) Prior to gastrulation, btk is expressed ubiquitously, after which it is restricted to hematopoietic precursors. (A) Zebrafish embryos were collected, fixed, and stained by in situ hybridization for anti-sense and sense btk transcript at several stages: shield, 14 hours post fertilization (hpf), 21 hpf, and 48 hpf. These data are representative of probes synthesized from two distinct regions of the btk open reading frame. (B-C) cDNA was purified from zebrafish embryos, and btk and actin was amplified by PCR. The results were run on a gel (B), and the bands were quantified by optical densitometry and then plotted as a ratio of btk to actin (C). (D) Attenuation of btk in zebrafish potentiates Wnt/β−catenin signaling. Injection of wnt8 mRNA into 1-cell stage zebrafish embryos causes anterior truncation in zebrafish by 24 hours post fertilization. Embryos co-injected with wnt8 mRNA (1.5 pg) and the second btk morpholino (denoted concentration) exhibited dose-dependent increases in the number and severity of anterior truncation phenotypes relative to co-injection with control morpholino (D). Note that these data are representative of four independent experiments. (E) To confirm that our btk morpholinos disrupted splicing, zebrafish were injected with 4 ng of morpholino and allowed to develop for 24 hours. At this time, genomic DNA was harvested and the indicated splice sites were amplified by PCR. btk morpholino 1 causes the formation of a second, smaller product (arrow), whereas btk morpholino 2 causes degradation of btk mRNA.

NormalSmallEyeHeadlessDorsalizedRadialized

Supplemental Figure 3. Protein Interaction Networks for BTK and CDC73.

A

B

BLNK PLCG1

VAV1

LYN WAS

GRB2

RNPS1

CBL

TXK

PLCG2

LRRC55MAP3K4

EIF3B EIF3F

EIF3C

ANKRD54

TRIM21

ANP32A

BTK

VPS53

hCG_15200

DPYSL5

HSPA1A

SRRM2 LOC650788

PRPF8

NUDT21

HTATSF1

ARID3A

SF3B4

ASCC3L1SNRPB2

PTX3

SET

SF3B2

WDR61

GTF2I

SSRP1

PAF1

CDC73

SH3BP5

FKBP5

NOLC1

CSNK2A1

CDC37

SUPT16H

TCOF1

EFHC1

UBR4

FAS

HIST1H2AE GNB2L1

PTGER1

L1TD1

SSRP1

JUP

CTR9

LEO1

DSG1

PAF1

CDC73

WDR61

DSC1

LYN

TXK

VAV1

PLCG1

BLNK

SF3B2

SNRPB2

PRPF8

HTATSF1

SNRPD2SF3B4

ASCC3L1

BTK

NUDT21

TCF7L2CTNNB1

PYGO1

LEF1

TCEB3

CSNK2A1

SUPT16H

EIF3B

ARID3A

EIF3C

FAS

SH3BP5

EIF3F

GTF2I

GRB2 NOLC1

PLCG2WAS

TCOF1

FKBP5

CBL

CDC37

PIP5K1A

SFRS3

CPSF1

CPSF2

SIT1

BCL9

Supplemental Figure 3. Protein interaction networks for BTK and CDC73. Proteins (nodes) are represented as circles (bait) or squares (prey) and the relationship between proteins (edges) are denoted by the lines between the nodes. This protein-protein interaction network (PIN) for BTK and CDC73 generated with data from the literature (red edges), and primary affinity purification, mass spectrometry data (blue edges). The width of the blue edge is directly correlated to the total number of peptides identified in three independent affinity purification experiments. (A) This PIN represents the total number of proteins that co-purified with BTK from multiple pull-downs from HEK293T cells. BTK interacts with the Wnt/β−catenin regulators CDC73, PAF1, and CSNK2A1. Red nodes represent proteins that have been reported previously to have physical linkage with Wnt/β−catenin signaling downstream of the β−catenin destruction complex. (B) This PIN represents selected proteins that co-purified in multiple independent experiments with either BTK or CDC73 from HEK293T cells. Proteins were chosen based on whether they interacted with both CDC73 and BTK (shaded in blue) or have been previously implicated in Wnt/β-catenin signaling and/or transcriptional elongation. This PIN demonstrates the physical relationship of BTK and CDC73 to the Wnt/β−catenin pathway.

0

30

60

90

Cnt

l

AX

IN1/

2

CTN

NB

1

CD

C73

#1

CD

C73

#2

siRNA:

Fold

β-c

aten

in R

epor

ter RKO cell

VehicleWNT3A

0

25

50

75

Cnt

l

AX

IN1/

2

CTN

NB

1

CD

C73

#1

CD

C73

#2

siRNA:

Fold

β-c

aten

in R

epor

ter HEK293T cell

VehicleWNT3A

A B

Supplemental Figure 4. Context-dependent regulation of Wnt/β−catenin signaling by CDC73. RKO B cells (A) or HEK293T cells (B) stably expressing a β−catenin activated reporter (BAR) upstream of firefly luciferase and expressing a constitutive reporter that drives renilla luciferase were transiently transfected with the indicated siRNA oligonucleotides. 48 hours after transfection, the cells were treated with either WNT3A- or control-conditioned medium. The following day, firefly luciferase was quantified, normalized to that of renilla luciferase, and all data were plotted as fold change over activity in untreated control siRNA-treated cells. Error bars represent standard deviation from the mean for 3 replicates. This data is representative of three independent experiments.

Supplemental Table 1. Sequences of siRNA sense strands.

Gene Symbol Sequence (5’>3’) Company

Control Negative control 1 Ambion

AXIN1 GGUGUUGGCAUUAAAGGUGdTdT Invitrogen

AXIN2 GGGAGAAAUGCGUGGAUACdTdT Invitrogen

CTNNB1 GGUGGUGGUUAAUAAGGCUdTdT Invitrogen

BTK #1 CCAGUGAAAUGGAGCAAAUdTdT Invitrogen

BTK #2 CCCUUAUCCCUUCCAGGUUdTdT Invitrogen

BTK #3 GCCAAUGAAUGCAAAUGAUdTdT Invitrogen

CDC73 #1 UUUGUAAGAUAGUUGUUCGUGdTdT Invitrogen

CDC73 #2 CAGCGAUCUACUCAAGUCAAAdTdT Invitrogen

Ctnnb1 CUGUCUGUCUGCUCUAGCAdTdT Invitrogen

Btk517 GCTGAGAAAGCGCTGGATTdTdT Invitrogen

Btk1012 GCACATGACTCGAAGTCAAdTdT Invitrogen

Btk1651 GCTGCTTGAGATGTGCAAAdTdT Invitrogen

Table S2. Descriptions of supplementary database files. Database

# Title File Name

(size in KB) Description

1 Small molecule screen in HT22 cells

2000230s1.xls (837)

Raw data for each small molecule that we screened is listed along with an accompanying Simplified Molecular Input Line Entry Specification String (SMILES), concentration, and source. The data are presented in order of decreasing effect on WNT3A activation.

2 Raw data of siRNA screen in RKO cells

2000230s2.xls (989)

Data from distinct pools of siRNAs targeting 2,518 genes are listed in rows. Each pool was screened in quadruplicate, and mean and median effect of the pool on WNT3A-mediated activation of a β-catenin activated reporter is listed.

3 Median reporter activity of all genes in the siRNA screen

2000230s3.xls (173)

The effect on WNT3A-mediated β-catenin activated reporter activity for each gene was listed as an average of the median reporter activity of all individual pools.

4 Hit list for small molecule screen

2000230s4.xls (36)

Small molecules that increased BAR activity greater than 1.5-fold were listed with their accompanying SMILES string, and fold-change over WNT3A alone.

5 Hit list for siRNA screen

2000230s5.xls (233)

Targets of siRNA that increased BAR activity (negative regulators of Wnt/β-catenin signaling) are highlighted in red and targets that decreased BAR activity (positive contributors to Wnt/β-catenin signaling) are highlighted in green.

6 Binary generated from STITCH search of small molecule screen hits

2000230s6.xls (124)

The results from a STITCH search of the hits in the chemical screen are presented as a binary in which the contents of the adjacent columns are direct interactors.

7 Binary generated from STRING search of siRNA screen hits

2000230s7.xls (398)

The results from a STRING search of the hits in the siRNA screen are presented as a binary in which the contents of the adjacent columns are direct interactors.

8 List of randomly chosen genes that did not hit in the siRNA screen

2000230s8.xls (23)

A random list of genes was chosen as a negative control for association in the bioinformatic overlap. The effect on WNT3A-mediated β-catenin activated reporter activity for each gene was listed as an average of the median reporter activity of all individual pools.

9 Binary generated from STRING search of siRNA screen non-hits

2000230s9.xls (81)

The results from a STRING search of the random list of genes chosen from the siRNA screen are presented as a binary in which the contents of the adjacent columns are direct interactors.

10 List of overlaps between small molecule STITCH search and siRNA STRING search

2000230s10.xls (37)

Presented is a list of 108 protein targets that interact with a small molecule screen hit and interact with a siRNA screen hit. The 108 proteins are grouped in three ways: (1) proteins that interact with a small molecule screen hit and that are among the 445 siRNA screen hits; (2) proteins that interact with a small molecule screen hit and were not present in the siRNA screen; and (3) proteins that interact with a small molecule screen hit and did not hit in the siRNA screen.

11 List of overlaps between small molecule STITCH search and a STRING search of siRNA non-hits

2000230s11.xls (17)

Presented is a short list of proteins that interact with a small molecule screen hit and interact with one of the proteins present in a random list of genes present in our siRNA screen.

12 All attribute files and the Cytoscape binary utilized to generate Fig. 2 and Supplementary Database 13

2000230s12.xls (853)

This Excel file has four worksheets, which each contain a binary used in the construction of the cytoscape visualization of the bioinformatic overlap of the small molecule and siRNA screens. The first worksheet (ScoreAttribute) is an attribute file that shows whether a specific protein or compound increased, decreased, or did not affect BAR. The second worksheet (CompoundAttribute) is a binary that was used as an attribute file to specify which nodes in the Cytoscape file are compounds. The third worksheet (CombinedBinary) is the binary containing all the interaction data present in supplementary databases 6 and 7. Finally, the fourth worksheet (SRI_STI_Overlap_Attribute) is an attribute file used to group interactions according to whether they originated from STITCH or from STRING.

13 Cytoscape file showing all siRNA and small molecule screen hits and their overlap

2000230s13.cys (285)

This Cytoscape interactome is the file presented in fig. S1.

14 Cytoscape file for BTK protein-protein interaction map

2000230s14.cys (22)

This Cytoscape interactome is the file presented in fig. S3A.

15 Cytoscape file for CDC73 and BTK protein-protein interaction map

2000230s15.cys (25)

This Cytoscape interactome is the file presented in fig. S3B.

16 Affinity-purification-mass spectrometry data for BTK

2000230s16.xls (21)

Listed is the raw data from the BTK coimmunopurification mass spectrometry experiments. Prey are listed along the accompanying information showing the total number of peptides identified in all experiments, and the peptides identified in independent experiments (BTK, BTK-1, and BTK-2).

17 Affinity-purification-mass spectrometry data for CDC73

2000230s17.xls (20)

Listed is the raw data from the CDC73 coimmunopurification mass spectrometry experiments. Prey are listed along the accompanying information showing the total number of peptides identified in all experiments, and the peptides identified in independent experiments (eCDC73-Nalm6, GLUe_CDC73_B1, and GLUE_CDC73_D1).

18 Isolation of CDC73 peptides from 60 kD band from silver stain

2000230s18.xls (24)

Listed are the peptides identified from in-gel digestion and mass spectrometry analysis of the prominent 60 kD band observed on silver stains following CDC73 immunoprecipitation.