Figure 2

TGF-β1 downregulates VEGF-D expression in MRC-5 cells via a TGF-β receptor type I (TGF-βRI)-dependent, Smad3-independent mechanism. (A, B) MRC-5 cells were treated for 1 h with SB431542 (10 µmol/L), an inhibitor of activin receptor-like kinase (ALK)-5, followed by the addition of TGF-β1 (5 ng/mL) for the indicated time points. Equal amounts of protein from whole cell lysates were analyzed by Western blotting with antibodies against phospho-Smad3 and total Smad3 (A) and VEGF-D, α-SMA and β-actin (B). (C) Ratio of VEGF-D to β-actin density was expressed as the fold change relative to unstimulated control in (B). (D, E) MRC-5 cells were transfected with control siRNA (Ctrl) or Smad3 siRNA for 24 h, serum-starved for 24 h and then incubated in serum-free medium in the absence or presence of TGF-β1 (5 ng/mL) for 48 h. Equal amounts of protein from whole cell lysates were analyzed by Western blot with antibodies against VEGF-D, Smad3, α-SMA and β-actin (D). Ratio of VEGF-D to β-actin density was expressed as the fold-change relative to control siRNA alone (E). Data represent means ± SEM of three independent experiments. *P < 0.05, ***P < 0.001, NS, not significant, by one-way ANOVA.