Figure 6

Changes in protein expression during H₂O₂-induced conversion of ECs into myofibroblasts inhibited by the specific inhibitor of Smad3, SIS3. (a–d) Cytosol (CF) and nuclear (NF) fractions were obtained from non-treated ECs and EC treated with TGF-β1 (10 ng/ml) in the presence or absence of 5 μM SIS3 for 72 h, and expression of smad3 was analyzed. (a, c) Representative images from western blot experiments performed for detection of smad3 in the CF (a) and NF (c) from non-treated ECs and EC treated with TGF-β1 in the presence or absence of SIS3. (b and d) Densitometric analyses of the experiments shown in (a and c), respectively. Protein levels were normalized against tubulin and histone H3 for CF and NF, respectively, and data are expressed relative to non-treated condition (N=3). (e–j) ECs were incubated in the absence (−) or presence (+) of 10 μM H₂O₂ for 72 h and treated in the absence (−) or presence (+) of 5 μM SIS3 for 72 h, and then protein expression was analyzed. (e, g, and i) Representative images from western blot experiments performed for detection of the endothelial marker VE-cadherin (VE-cad) (e), fibrotic marker α-SMA (g) and ECM proteins fibronectin (FN) (i). Panels (f, h, and j) show densitometric analyses from several experiments, as shown in (e, g, and i), respectively. Protein levels were normalized against tubulin, and the data are expressed relative to the untreated (0 μM H₂O₂ in the absence of SIS3) condition. Statistical differences were assessed by a one-way analysis of variance (ANOVA) (Kruskal–Wallis) followed by Dunn’s post hoc test. **: P<0.01 against the untreated (0 μM H₂O₂ in the absence of SIS3) condition. NS: non-significant. Graph bars show the mean±s.d. (N=3–4).