Fig. 4

OVOL1 inhibits the TGF-β/SMAD signaling pathway and TGF-β-induced EMT. a Reporter assays for measuring the luciferase activity in HEK293T (left panel) or HepG2 cells (right panel) transfected with TGF-β-induced SMAD3/4-dependent CAGA-luc transcriptional reporter and indicated constructs for the ectopic expression (left) or depletion (right) of OVOL1. The results are expressed as mean ± SD. **0.001 < P < 0.01, ***0.0001 < P < 0.001. b PAI-1 and CTGF expression as detected by RT-qPCR in MDA-MB-231 cells with OVOL1 ectopic expression induced by Doxycycline (Dox). Cells were kept in the presence or absence of Dox for 2 days before serum starvation overnight and the treatment of TGF-β (1 ng/ml) for indicated time points. Statistical analyses were carried out at the indicated time points. The results are expressed as mean ± SD. *0.01 < P < 0.05, **0.001 < P < 0.01. c RT-qPCR detection of PAI-1 and CTGF expression in MCF10A-M2 cells upon the knockdown of OVOL1. The results are displayed as mean ± SD in technical triplicates. d Western blotting quantification of the phosphorylation of SMAD2 (p-SMAD2) and total SMAD2 (t-SMAD2) in MDA-MB-231 cells without (Mock) or with inducible OVOL1 ectopic expression (+Tet-ON OVOL1). Cells were treated without or with Doxycycline (Dox) for 2 days before serum starvation overnight and the stimulation of TGF-β (1 ng/ml) for indicated time points. To control for equal loading, GAPDH levels were analyzed. e The phosphorylation of SMAD2 (p-SMAD2) and total SMAD2 (t-SMAD2) quantified by western blotting in MCF10A-M2 cells with inducible OVOL1 depletion. Cells were kept in the presence or absence of Doxycycline (Dox) for 2 days before the stimulation of TGF-β (1 ng/ml) for indicated time points. f Western blotting analysis of changes in mesenchymal markers expression in MCF10A-M2 cells upon OVOL1 knockdown induced by Doxycycline (Dox). Cells were treated without or with Dox for 2 days before the stimulation of TGF-β (2.5 ng/ml) for another 2 days. To control for equal loading, GAPDH levels were analyzed. g Western blotting measurement of mesenchymal markers expression in MCF10A-M2 cells with the depletion of OVOL1. Cells were either not treated or treated with SB431542 (SB) for 2 days. To control for equal loading, Vinculin levels were analyzed. h Immunofluorescence detection of F-actin and DAPI staining of HaCaT cells upon OVOL1 knockdown induced by Doxycycline (Dox). Cells were treated without or with SB431542 (SB) for 2 h and kept in the presence or absence of Dox for 48 h