Fig. 3

Activin A increases fibronectin expression through ALK4-activated SMAD2/3-SMAD4 signaling in HTR8/SVneo cells. A, B and C, HTR8/SVneo cells were pretreated for 1 h with vehicle control dimethylsulfoxide (DMSO) or 10 μm SB431542 before treatment without (Ctrl) or with 50 ng/mL activin A for 20 and 40 minutes for P-SMAD2/SMAD2 and P-SMAD3/SMAD3 protein (A), 12 and 24 h for FN1 mRNA (B) or 24 and 48 h for fibronectin protein (C), examined by RT–qPCR or Western blot, respectively. D, E, and F, HTR8/SVneo cells were treated with or without 50 ng/mL activin A after transfection with 20 nM si-Ctrl or si-ALK4. The mRNA level of ALK4 (ACVR1B) was examined by TaqMan gene expression assay (D). The mRNA and protein levels of fibronectin were measured using RT–qPCR (E) and Western blot (F), respectively. G, H and I, HTR8/SVneo cells were treated with or without 50 ng/mL activin A after transfection with 20 nM si-Ctrl or si-SMAD4. The mRNA and protein levels of SMAD4 and fibronectin were measured by RT–qPCR (G and H) and Western blot (I), respectively. Quantitative results are expressed as the mean ± SEM of at least three independent experiments. ANOVA was used for grouped analyses. Groups without letters in common are significantly different from each other (P < 0.05). P values of primary interest are denoted as * < 0.05, ** < 0.01, and *** < 0.001