SMAD2 and STAT3 activation in human HCC tumors

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SUPPLEMENTAL FIGURE LEGENDS

Supplemental Figure 1: SMAD2 and STAT3 activation in human HCC tumors. (A) Heatmap of SMAD2 and SKI expression in human hepatocellular carcinoma (HCC) tissue of 80 patients. Data were obtained from the GEO database (GEO ID: GPL5474) and analyzed by Cox regression analysis followed by Ingenuity Pathway Analysis (IPA); “red” indicates upregulation, “green” represents downregulation.

(B) PAI-1 transcription in non-malignant hepatic tissue

versus matching HCC tumor tissue in an independent set of HCC patients (n=27). mRNA was quantified by qRT-PCR and data were normalized for GAPDH. Data are shown as fold increase in PAI-1 transcription in tumor tissue versus matching non-malignant tissue. The red line represents the median.

(C) Kaplan-Meier survival analysis of an independent set of HCC

patients (n=80) with tumors immunohistochemically positive or negative for intranuclear pSer465/467SMAD2.

(D) Allred scoring system based correlation analysis of human HCC tumors

(n=116) immunohistochemically analyzed for pSer465/467SMAD2 and pTyr705STAT3 . (E) Immunofluorescence analysis for intracellular pSer465/467SMAD2/pTyr705STAT3 co-positivity in human HCC tumors tissue.

Supplemental Figure 2: TGF-β-induced STAT3-activation in human liver cancer cells. (A) Immunofluorescence analysis for TGF-β1-induced nuclear translocation and accumulation of pTyr705STAT3 in HepG2 and Hep3B cells. (B) Immunoblot analysis of cell lysates of human liver cancer cell lines treated with TGF-β1 for 3, 6, 9 and 12 hours. The top panel shows the analysis for SNU449 and SNU398 cells, and the middle and bottom panel shows the immunoblot analysis for HepG2 and Hep3B cells, and their SMAD2-knockdown clones (experiments were performed in parallel with the parental cell lines. (C) Immunoblot analysis of cell lysates for STAT3. Using

two distinct STAT3 targeted shRNA clones (cl.1 and cl.2), polyclonal cell populations of HepG2 and Hep3B cells were generated by lentiviral transduction. Immunoblot analysis confirmed

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successful silencing of STAT3. β-actin was used as a loading control. (D) Immunofluorescence

analysis for EMT-related proteins vimentin (red) and E-cadherin (green), and nuclear DAPI stain (blue) in parental versus polyclonal STAT3 knockdown HepG2 cells; in contrast to Hep3B cells, no vimentin was detected in HepG2 cells. (E) qRT-PCR analysis for TGF-β1-induced transcription of EMT-markers in parental HepG2 and Hep3B cells, and their polyclonal STAT3knockdown derivatives.

Supplemental Figure 3: Restoration of TGF-β tumor suppressor function. (A) Liver tumor cell viability analysis after 48h of TGF-β1 stimulation (10 ng/mL) +/- STAT3-inhibitor STATTIC (5 μM) using MTT assay. (B) TUNEL-assay of parental and polyclonal STAT3-knockdown liver tumor cells treated with TGF-β1 (10 ng/mL). The propapototic DR4/5 ligand TRAIL (100 ng/mL) was used as a control. Quantification of apoptotic cells was performed in 3 high-power fields (mean +/- SD).

(C) Immunoblot analysis for cell cycle regulatory proteins in human liver tumor

cells stimulated with TGF-β1 +/- STAT3 inhibitor STATTIC (5 μM).

Supplemental Figure 4: TGF-β-mediated activation of c-KIT/STAT3-signaling. (A) and (B) JAK1/2-dependence of TGF-β-induced STAT3-activation analyzed by immunoblot and immunofluorescence analysis for pTyr705STAT3 following stimulation with TGF-β1 +/- JAK1inhibitor (0.1 μM). The bar graphs below the immunofluorescence images show the number of cells in the immunofluorescence analysis with intranuclear pTyr705STAT3 accumulation (mean +/SEM of 5 high-power fields). (C) qRT-PCR analysis for TGF-β1 induced SCF-transcription in parental and SMAD2-knockdown HCC cells; results were normalized for GAPDH. (D) c-KITdependence of TGF-β-induced STAT3 activation analyzed by immunoblot analysis for pTyr705STAT3 following stimulation with TGF-β1 +/- c-KIT inhibitor ISCK03 (5 μM). (E) ELISA for

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SCF in supernatant of parental and polyclonal SCF-knockdown HepG2 and Hep3B cells following 12 hours of TGF-β1 stimulation (mean +/- SD). (F) Schematic representation of the SCF promoter with putative SMAD Binding Elements (SBE) shown as boxes (white boxes = AGAC) with their relative position to the start codon. ChIP primer location is shown below with their relative positions to the SCF start codon and the PCR product size. The full-length and the three truncated SCF promoter fragments that were cloned into the luciferase expression vector are shown below. (G) TGF-β1-induced (10 ng/mL, 24h-stimulation) luciferase activity using SCF promoter-regulated luciferase expression plasmids transfected into liver tumor cells.

Supplemental Figure 5: SCF expression in human HCC tumors. (A) Kaplan-Meier survival analysis of HCC patients (n=80) with tumors immunohistochemically positive or negative for cytosolic SCF.

Supplemental Figure 6: SCF/STAT3-mediated TGF-β-autoregulation. (A) ELISA for TGF-β1 in the supernatant of SCF treated parental and STAT3-knockdown HepG2 and Hep3B cells. The results are shown as total amount of SCF in cell supernatant per mg protein lysates per 48 hours. (B) Schematic of the TGFB promoter with putative STAT3 binding sites shown as grey boxes with their relative position to the start codon. ChIP primer locations are shown with their relative positions to the SCF start codon and the PCR product sizes indicated below. Below, the three TGFB gene fragments are shown with their indicated relative position to the TGFB start codon,

and their position within the luciferase expression vectors. (C) TGF-β1-induced (10 ng/mL, 24h) luciferase activity using full-length SCF-promoter-regulated luciferase expression plasmids transfected into parental and SCF knockdown liver tumor cell lines.

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Supplemental Figure 7: Preliminary studies. (A) Immunoblot analysis of protein lysates of HepG2 cells treated with TGF-β1 (10 ng/mL) in the presence or absence of inhibitors for JAK1, p38MAPK, Src, ERK1/2 and EGFR. (B) Following cytokine array analysis (data not shown, available upon request), IL-6 dependence of TGF-β1-induced STAT3-phosphorylation was evaluated. HepG2 cells were transfected with three different siRNA against the α-subunit (gp80) of the IL-6 receptor. Subsequently, untransfected and transfected HepG2 cells were treated with TGF-β1 (10 ng/mL). Following protein isolation, immunoblot analysis of protein lysates was performed for gp80 to confirm successful gp80 knockdown and for pTyr705STAT3; β-actin was chosen as a loading control.

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Supplemental Figure 1

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B) Tumor vs. normal tissue [fold increase in mRNA]

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Advanced stage HCC:

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Supplemental Figure 2

A) DAPI

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Supplemental Figure 2 C)

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Supplemental Figure 2

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