SUPPLEMENTARY INFORMATION

1. Supplementary Figures

Figure S1. MIR506 inhibits proliferation in PDAC cells. (A) MDA-PATC53, MDA-PATC124, and MDA-

PATC148 cells were transfected with MIRctrl or MIR506 for 72 h. Cells were fixed for immunofluorescent

staining with anti-MKI67 antibody and visualized with a fluorescence microscope. The percentage of

MKI67-positive cells was determined. Scale bar: 100 µm. (B) Transfected cells were seeded in a 6-well

plate (800 cells/well) and kept in culture for 7 days. Colony formation was analyzed by staining the cells

with 0.1% crystal violet. Each bar represents the mean ± SD of at least of 3 independent experiments.

**P<0.01.

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Figure S2. MIR506 inhibits proliferation of PDAC cells by inducing cell cycle arrest. (A) MDA-PATC53,

MDA-PATC124, and MDA-PATC148 cells were transfected with MIRctrl or MIR506 for 72 h. BrdU-

pretreated cells were stained with FITC-conjugated anti-BrdU antibody and 7-AAD for flow cytometry

analysis. (B) Fixed cells were stained with PI and analyzed by flow cytometry for DNA content. Each bar

represents the mean ± SD of at least of 3 independent experiments.

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Figure S3. MIR506 inhibits proliferation of PDAC cells by directly targeting CDK4 and CDK6. (A) MDA-

PATC53 cells were transfected with MIRctrl or MIR506. Whole-cell extracts were subjected to

immunoblotting with anti-CDK4 and -CDK6 antibodies. (B) The expression level of CDK4 and CDK6 was

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detected by qRT-PCR and normalized to the endogenous control GAPDH. (C) Potential MIR506 binding

sites were predicted in the 3’-UTR of CDK4 and CDK6 by TargetScan. (D) Luciferase reporter assay

showed that MIR506 directly targeted the 3’-UTR of CDK4 and CDK6. MDA-PATC53 cells were

cotransfected with wild-type or mutant CDK4 or CDK6 3’-UTR-luciferase reporter and MIRctrl or MIR506

mimic for 72 h before analysis. The firefly and renilla luciferase activities were measured using a Dual

Luciferase Reporter Assay System. Data represent the mean ± SD of at least 3 independent experiments.

NS, not significant; *P<0.05; **P<0.01.

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Figure S4. Knockdown efficiency of ATG5 and ATG7. Two essential autophagy genes, ATG5 or ATG7, were knocked down by shRNA. (A) and (B) show the results of knockdown efficacy for ATG5 and ATG7, respectively, at the protein level measured by western blotting.

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Figure S5. MIR506 induces autophagic flux in PDAC cells. MDA-PATC53, MDA-PATC124, and MDA-

PATC148 cells were transfected with MIRctrl or MIR506 for 72 h in the absence or presence of 3-MA (5

mM). Cells were stained with 1 mg/mL AO and analyzed by flow cytometry. Each bar represents the

mean ± SD of at least of 3 independent experiments. **P<0.01.

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Figure S6. MIR506 induces autophagic flux in PDAC cells. MDA-PATC53, MDA-PATC124, and MDA-

PATC148 cells were transfected with MIRctrl or MIR506 for 72 h in the absence or presence of the

vacuolar-type H+-ATPase inhibitor Baf-A (10 nM). (A) Whole-cell extracts were subjected to

immunoblotting with anti-LC3 and -SQSTM1 antibodies. (B) The relative fold-changes of LC3-II to ACTB

were quantified by densitometric analysis.

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Figure S7. MIR506 induces autophagy in a STAT3-dependent manner. MDA-PATC53, MDA-PATC124,

MDA-PATC148, MDA-PATC50, and MDA-PATC69 cells were transfected with MIRctrl or MIR506 for 72 h

and stained with 1 mg/mL AO. Cells were analyzed by flow cytometry. Each bar represents the mean ±

SD of at least 3 independent experiments. NS, not significant; **P<0.01.

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Figure S8. MIR506 induces autophagy through the STAT3 signaling pathway. MDA-PATC53, MDA-

PATC124, and MDA-PATC148 cells were treated with indicated concentrations (A) of STAT3 inhibitor

Stattic for the indicated times (B). Cells were stained with 1 mg/mL AO and analyzed by flow cytometry.

Each point represents the mean ± SD of at least of 3 independent experiments.

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Figure S9. MIR506 induces autophagy through the STAT3 signaling pathway. MDA-PATC53, MDA-

PATC124, and MDA-PATC148 cells were transfected with control siRNA (siR-ctrl) or 1 of 2 siRNAs against

STAT3 (siR-STAT3-1 or siR-STAT3-2) for 72 h. Cells were stained with 1 mg/mL AO and analyzed by flow

cytometry. Each bar represents the mean ± SD of at least of 3 independent experiments. **P<0.01.

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Figure S10. MIR506 induces autophagy through the STAT3 signaling pathway. MDA-PATC53 cells

overexpressing wild-type or Y705-mutated STAT3 were transfected with MIRctrl or MIR506 for 72 h.

Cells were stained with 1 mg/mL AO and analyzed by flow cytometry. Each bar represents the mean ±

SD of at least of 3 independent experiments. NS, not significant; **P<0.01.

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Figure S11. MIR506 induces autophagy through the STAT3-BCL2-BECN1 signaling pathway. MDA-PATC53

cells were transfected with control shRNA (shR-ctrl) or 1 of 2 shRNAs against BECN1 (shR-BECN1-1 or

shR-BECN-2). Cells were stained with 1 mg/mL AO and analyzed by flow cytometry. Each bar represents

the mean ± SD of at least of 3 independent experiments. NS, not significant; **P<0.01.

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Figure S12. MIR506 induces autophagy through the STAT3-BCL2-BECN1 signaling pathway. MDA-PATC53

cells transformed with empty vector (EV) or overexpressing BCL2 were transfected with MIRctrl or

MIR506 for 72 h. Cells were stained with 1 mg/mL AO and analyzed by flow cytometry. Each bar

represents the mean ± SD of at least of 3 independent experiments. NS: not significant; **P<0.01.

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2. Supplementary Tables

Table S1. Baseline demographic and clinicopathological characteristics of pancreatic adenocarcinoma

patients and their correlation with MIR506 expression.

CharacteristicNo. MIR506 expression

χ2 P-value(n=92) Low (n=46) High (n=46)

Gender 0.046 0.830

Male 57 28 (60.9%) 29 (63.0%)

Female 35 18 (39.1%) 17 (37.0%)

Age, years 0.179 0.672

≤60 38 18 (39.1%) 20 (43.5%)

>60 54 28 (60.9%) 26 (56.5%)

Tumor location 0.205 0.428

Head 64 31 (67.4%) 33 (71.7%)

Body/Tail 28 15 (32.6%) 13 (28.3%)

Tumor size, cm 0.049 0.825

≤4 61 31 (67.4%) 30 (65.2%)

>4 31 15 (32.6%) 16 (34.8%)

Tumor differentiation 1.087 0.581

Well 10 6 (13.0%) 4 (8.7%)

Moderate 56 29 (63.0%) 27 (58.7%)

Poor 26 11 (23.9%) 15 (32.6%)

Perineural invasion 0.747 0.388

Negative 58 27 (%) 31 (%)

Positive 34 19 (%) 15 (%)

Vascular invasion 0.522 0.470

Negative 69 33 (71.7%) 36 (78.3%)

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Positive 23 13 (28.3%) 10 (21.7%)

T status 14.33 <0.001**

T1-T2 40 11 (23.9%) 29 (63.0%)

T3-T4 52 35 (76.1%) 17 (37.0%)

N status 2.831 0.093

N0 40 16 (34.8%) 24 (52.2%)

N1 52 30 (65.2%) 22 (47.8%)

M status 4.358 0.037

M0 82 39 (84.8%) 43 (93.5%)

M1 10 7 (15.2%) 3 (6.5%)

TNM stage (AJCC) 9.946 0.002**

I-II 74 31 (67.4%) 43 (93.5%)

III-IV 18 15 (32.6%) 3 (6.5%)

TNM stage: TNM Classification of Malignant Tumors; T status: the size of the original (primary) tumor and nearby tissue

invaded; N status: nearby (regional) lymph nodes involved; M status: distant metastasis. ** P< 0.01.

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Table S2. Antibodies used for western blot and immunofluorescence.

Antibody Isotype Catalog Manufacturer

anti-LGALS1 rabbit ab25138 Abcam, MA, USA

anti-LGALS3 rabbit ab53082 Abcam, MA, USA

anti-LC3B rabbit 3868 Cell Signaling Technology, MA, USA

anti-STAT3 rabbit 12640 Cell Signaling Technology, MA, USA

anti-phospho-STAT3 (Tyr705) rabbit 9145 Cell Signaling Technology, MA, USA

anti-CASP3/caspase-3 rabbit sc-7148 Santa Cruz Biotechnology, CA, USA

anti-CASP9/caspase-9 mouse sc-70505 Santa Cruz Biotechnology, CA, USA

anti-BECN1/Beclin 1 rabbit sc-11427 Santa Cruz Biotechnology, CA, USA

anti-BCL2/Bcl-2 mouse sc-7382 Santa Cruz Biotechnology, CA, USA

anti-SQSTM1 mouse sc-28359 Santa Cruz Biotechnology, CA, USA

anti-MKI67/Ki-67 rabbit sc-15402 Santa Cruz Biotechnology, CA, USA

anti-TUBB/β-tubulin rabbit sc-9104 Santa Cruz Biotechnology, CA, USA

anti-ACTB/β-actin mouse sc-47778 Santa Cruz Biotechnology, CA, USA

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