Fig. 3: Loss of SNAI1 dysregulates TGFβ signaling and EMT pathways in MDA-MB-231 cells.

A Representative phase contrast images from three biological repeats showing the morphology of MDA-MB-231-WT, CS16 and CS16 SNAI1-GFP#7 cells. Scale bars = 100 μm. B Protein expression levels of SNAI1 and of the EMT transcription factors ZEB1, SNAI2, of the epithelial protein CDH1 and of the mesenchymal proteins FN1, VIM in MDA-MB-231-WT, CS16 and CS16 SNAI1-GFP#7 cells. β-Actin serves as loading control. Stars indicate the specific protein bands; upper panel: stars correspond to SNAI1-GFP (top) and SNAI1 (bottom). C Protein expression levels of SMAD2/3, pSMAD2/3, TGFBRI and TGFBRII in MDA-MB-231-WT and SNAI1-KOs stimulated with 5 ng/ml of TGFβ1 for the indicated time periods. HP95 serves as loading control. Stars indicate the bands corresponding to pSMAD2 and pSMAD3. D RT-qPCR analysis of TGFBRI, TGFBRII mRNA levels in MDA-MB-231-WT and SNAI1-KO cells stimulated with 5 ng/ml of TGFβ1 for the indicated time periods. E Protein expression levels of PAI-1 in MDA-MB-231-WT and SNAI1-KOs stimulated with 5 ng/ml of TGFβ1 for the indicated time periods. HP95 serves as loading control. Immunoblots in B, C and E are representative of three biological replicates along with molecular mass markers in kDa. F RT-qPCR analysis of SMAD7 mRNA levels in MDA-MB-231-WT and SNAI1-KOs stimulated with 5 ng/ml of TGFβ1 for the indicated time periods. Values in D and F represent relative mRNA expression normalized to GAPDH. Data in D and F are presented as mean values of three biological replicates ± SEM, each in technical triplicates and p-values are shown based on two-tailed unpaired Student’s t-test. G Cell viability assay of MDA-MB-231-WT, SNAI1-KOs and ZR-75-1 cells in increasing concentrations of paclitaxel and doxorubicin as measured by PrestoBlue after 48 hours. The data are shown as mean values of three biological replicates ± SEM, each in technical triplicates. p-values *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.