Figure 6. Suppressing Smad7 protein expression with Notch inhibitor N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester facilitates epithelial–mesenchymal transition and growth arrest induced by transforming growth factor-beta. A: Western blot analysis of Smad 2/3 phosphorylation. Left planes: Transforming growth factor-beta (TGFβ1) time-dependently induces Smad2/3 phosphorylation in P1 cells. Right planes: P1 cells were treated with 10 ng/ml TGFβ1 for 30 and 40 min as positive control. P0 and P1 cells were pretreated with 10 µM N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (DMSO) vehicle for 48 h and then stimulated with TGFβ1 for 30 and 40 min, respectively. Cells were harvested and subjected to western blot analysis with phosphospecific antibodies of Smad2 and Smad3. The total protein levels of Smad2 and Smad3 were estimated by reprobing the membranes with total Smad2 and Smad3 antibody shown in the panels below. B: P0 LSCs pretreated with DAPT facilitates mesenchymal marker expression induced by TGFβ1. Representative blots (left panels) and densitometric analysis with standard deviation (SD; right columns) of three independent experiments are shown. *p<0.05 versus DMSO/TGFβ1-treated cells. C: P0 LSCs pretreated with DAPT facilitates growth arrest induced by TGFβ1. Cells were pretreated with DMSO or DAPT for 48 h and then treated with TGFβ1 for further 24 h. Subsequently, total RNA was extracted for determining Ki67 mRNA with quantitative real-time PCR (qPCR) assay as described in Figure 2C. Data represent three independent experiments. *p<0.002 versus DMSO/TGFβ1-treated cells.