FIG. 3.

In vitro cross-talk between the TGFβ and BMP signaling pathways. (A) Treatment of DM cells undergoing osteogenic differentiation with SB431542 leads to effective inhibition of TGFβ signaling as demonstrated by reduced pSmad2/3 at all time points. Quantification of electrophoretic bands (lower panel) is as already mentioned. Membranes were stripped and reprobed with α−tubulin antibody to control for equal loading. Total Smad2/3 proteins were detected by specific anti-pan Smad2/3 antibody. (B) SB431542 treatment inhibits TGFβ signaling also in POb cells undergoing osteogenic differentiation as indicated by reduced endogenous pSmad2/3 at all time points. Lower panel shows quantification of electrophoretic bands. (C) SB431542 treatment of DM cells undergoing osteogenic differentiation increases significantly endogenous pSmad1/5, at 24 and 48 h, signifying increased activation of BMP signaling. Quantification of pSmad1/5 electrophoretic bands (lower panel). Membranes were stripped and reprobed with α−tubulin antibody to control for equal loading. Total Smad1/5 proteins were detected by specific anti-pan Smad5 antibody. (D) SB431542 treatment leads to increase of endogenous pSmad1/5 also in POb cells. Quantification of pSmad1/5 electrophoretic bands (lower panel). Representative blots of three independent experiments. (E) qPCR analysis reveals upregulation of the pSmad1/5 target gene, Id2 in DM cells treated with SB431542 supporting further the activation of BMP signaling. (F) Similarly, an upregulation of Id2 expression is observed also in SB431542-treated POb cells. *p ≤ 0.05. BMP, bone morphogenetic protein. Color images available online at www.liebertpub.com/tea