Fig. 4

SMAD2 and SMAD3 mediate the GDF-11-induced downregulation of CGB expression. A BeWo cells were treated with 10 ng/mL GDF-11 (G11) for 10, 30, or 60 min. The levels of phosphorylated and total forms of SMAD2 and SMAD3 were determined by western blot (upper panel). The levels of phosphorylated forms of SMAD1/5/8 and the total form of SMAD1 were determined by western blot (lower panel). Cell lysate obtained from BeWo cells treated with 10 ng/mL BMP-4 (B4) for 60 min was used as a positive control. B JEG-3 cells were treated with 10 ng/mL GDF-11 (G11) for 10, 30, or 60 min. The levels of phosphorylated and total forms of SMAD2 were determined by western blot. C-E BeWo (C), JEG-3 (D), and primary CTB (E) cells were transfected with 50 nM (for BeWo and JEG-3) or 100 nM (for primary CTB) control siRNA (si-Ctrl) or SMAD4 siRNA (si-SMAD4) for 48 h, and then treated with 10 ng/mL GDF-11 (G11) every 24 h for 48 h. The protein levels of CGB and SMAD4 were examined by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. The values without a common letter are significantly different (p < 0.05)