znf217-overexpressing stable cells, znf217-1 and znf217-2...
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Supplementary Figure S1. ZNF217-overexpressing stable cells, ZNF217-1 and ZNF217-2 (3), display increased cell proliferation/viability. (A) Western-blot analysis of ZNF217. (B) Cell proliferation/viability was analyzed by counting the number of cells after 0, 1, 3, 5 and 7 days of culture. Results are expressed as means ± SD from three independent experiments. (C) Western-blot analysis of ZNF217 in cells transfected or not (NT) with either scrambled RNA, siRNA-A ZNF217 or siRNA-B ZNF217. Cell proliferation/viability was analyzed by counting the number of cells after 0, 1, 3, 5 and 7 days of culture. Results are expressed as means ± SD from three independent experiments. ***P < 0.001 (Student t-test).
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Supplementary Figure S2. ZNF217 silencing inhibits migration of ZNF217-2 cells. Confluent monolayers of ZNF217-2 cells transfected or not (NT) with either scrambled RNA, siRNA-A ZNF217 or siRNA-B ZNF217 were wounded by scratching and photographed at 0, 15 or 24 hours. Images (×2.5 magnification) are representative of at least three independent experiments.
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Supplementary Figure S3. Overview of deregulated expression of cell adhesion molecules in MDA-MB-231-ZNF217 cells. The KEGG pathway map was used. Genes in red represent deregulated genes in the total population of MDA-MB-231-ZNF217 cells compared with the control cell population (|FC| ≥ 1.7 in the two independent cell-culture replicates).
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Supplementary Figure S4. ZNF217 induces mammosphere formation. (A) MDA-MB-231-pcDNA6, ZNF217-1, ZNF217-2 cells and (B) population of MDA-MB-231-pcDNA6 and MDA-MB-231-ZNF217 cells were grown in non-adherent culture conditions and after 7 days mammospheres were counted (mean ± SD of three independent experiments). *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test).
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Supplementary Figure S5. ZNF217 induces invasion and EMT in HMLE cells. (A) Representative images of cell invasion Matrigel assay from at least three independent experiments. Scale bar, 400 µm. (B) Representative images of HMLE-pcDNA6 and HMLE-ZNF217 cell morphology at ×10 magnification. (C) Western-blot analysis of ZNF217, epithelial markers and mesenchymal markers. (D) Immunofluorescence acquisition showing E-cadherin and actin subcellular localization in HMLE-pcDNA6 cells (treated or not for 48 hours with 5 ng/ml of TGF-β1) and in HMLE-ZNF217 cells. Scale bar, 25 µm.
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Supplementary Figure S6. Inhibition of the TGF-β signaling pathway reverses ZNF217-induced EMT in HMLE cells. (A) Immunofluorescence acquisition showing E-cadherin and actin subcellular localization in HMLE-pcDNA6 and HMLE-ZNF217 cells treated (for 48 h) or not with 10 nM of SB431542. (B) Western-blot analysis of Smad4 in HMLE-ZNF217 cells transfected or not with either scrambled RNA, siRNA-A Smad4 or siRNA-B Smad4. Immunofluorescence acquisition shows subcellular localization of E-cadherin in these cells Scale bar, 25 µm.
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Supplementary Figure S7. (A) Gene expression of TGF-β family members measured by RTQ-PCR (mean ± SD of three independent experiments) in HMLE-ZNF217 and HMLE-pcDNA6 cells. *P < 0.05 and **P < 0.01 (Student t-test). (B) ChIP assays using anti-ZNF217 antibody or human IgG as a control on TGFB2 and TGFB3 promoter regions. Immunoprecipitated ChIP DNA was analyzed by PCR using ZNF217 binding-site-specific primers (Supplementary Table S3).
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Supplementary Figure S8. Overview of deregulation of the TGF-β signaling pathway in MDA-MB-231-ZNF217 cells. The KEGG pathway map was used. Genes in red represent deregulated genes in the total population of MDA-MB-231-ZNF217 cells compared with the control cell population (|FC| ≥ 1.7 in the two independent cell-culture replicates).
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Supplementary Figure S9. Quantification of protein expression of ZNF217, epithelial markers (E-cadherin, α-catenin, β-catenin and occludin) and mesenchymal markers (vimentin and N-cadherin) in MCF10A-pcDNA6 cells and MCF10A-ZNF217 cells (Fig. 5D). Histograms represent quantification of Western-blot signals for protein expression normalized to tubulin expression (mean ± SD of at least three independent experiments). **P < 0.01 and ***P < 0.001 (Student t-test).
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Vimentin N-cadherin
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Vimentin N-cadherin
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Supplementary Figure S10. (A) In addition to Figure 6B, Western-blot analysis (72 h post-transfection) of Smad4 in cells transfected or not with either scrambled RNA, siRNA-A Smad4 or siRNA-B Smad4. (B) In addition to Figure 6C, histograms represent the measurement of the distance between edges of the wound in at least three independent experiments. ***P < 0.001 (Student t-test).