Fig. 3.

Activation of the SMAD signaling pathway is not involved in TGF-β1-induced kisspeptin expression in HTR-8/SVneo cells. A Cells were treated with 5 ng/mL TGF-β1 for 10, 30, and 60 min. The levels of phosphorylated and total forms of SMAD2 and SMAD3 were determined by western blot. B and C Cells were transfected with 50 nM control siRNA (si-Ctrl) or SMAD4 siRNA (si-SMAD4) for 48 h, and then treated with 5 ng/mL TGF-β1 for 24 h. The kisspeptin, Cx43, and SMAD4 mRNA levels (B) and protein levels of kisspeptin and SMAD4 (C) were examined by RT-qPCR and western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values that are statistically different from one another (p < 0.05) are indicated by different letters