Figure 3

Smad3 regulates myofibroblast transdifferentiation and binds to BRD4 during TGFβ stimulation. (a) hDFs were electroporated with control or Smad3 siRNA. After 72 h incubation, cells were stimulated with TGFβ (10 ng/ml) for 48 h before analysis. Top panel, whole-cell extracts from hDF were analyzed for Smad3 content by western blotting. GAPDH was used as a loading control. Bottom panel, Smad3 mRNA expression was analyzed by quantitative real-time PCR (qRT-PCR). Immunofluoresence and qRT-PCR experiments were repeated at least three times. Western blotting was repeated twice. (b) hDF cells were fixed and immunostained for αSMA (green) and phalloidin for filamentous actin (red). Myofibroblasts were quantified in three separate experiments. Data presented are the mean of three experiments. (c) qRT-PCR of myofibroblast and ECM gene expression. Data in panels (a−c) are presented as mean±S.E.M. **P<0.05 versus −TGFβ; ^#P<0.05 versus vehicle+TGFβ . (d) Immunofluoresence staining for Smad2/3, phospho-Smad2/3, BRD4 and CDK9 in hDF cells incubated with or without TGFβ (10 ng/ml) for 24 h. DAPI was used to stain nuclei. Scale bar represents 100 microns. Immunofluoresence experiments were repeated twice and similar results were observed. (e) hDFs were treated with TGFβ (10 ng/ml) for 24 h. Equal amount of cell lysates were immunoprecipitated with anti-Smad3 antibody and subjected to SID-SRM-MS analysis of Smad3 and BRD4 protein levels. Data are presented as the mean ratio of native to SIS peptides or as BRD4 signal normalized to Smad3. (f) hDF cells were treated with TGFβ (10 ng/ml) for 24 h and cell lysates were immunoprecipitated with an anti-BRD4 antibody before being subjected to SID-SRM-MS analysis for BRD4, Smad3 and CDK9 proteins. Data are presented as the mean ratio of native to SIS peptide. All immunoprecipitation experiments were repeated three times. Bar graphs represent mean±S.E.M. *P<0.05 versus –TGFβ