Fig. 3

TGF-β1 induces the SMAD1/5/8 and SMAD2/3 signaling through the ALK5-containing receptor complex. A and B C3H10T1/2 cells were treated with ligand (TGF-β1 (0.1 nM) and/or BMP4 (0.1 nM)) in combination with inhibitors (SB-431542 (SB, 10 µM) or dorsomorphin (DM, 10 µM)) as indicated. The cells were subjected to Western blots (A) to evaluate changes in phosphorylated SMADs or BRE-driven luciferase reporter activity (B) to quantify SMAD1/5/8 response. Reporter activities are shown as means ± SD. ***P < 0.001 compared to the control within each group, two-way ANOVA followed by multiple comparison test. C-E Real-time PCR quantification of the TGF-β signaling-related receptor transcripts in C3H10T1/2 cells (C), mBM-MSCs (D) and hBM-MSCs (E). F and G The gene encoding Alk3 or Alk5 in C3H10T1/2 cells was knocked down by corresponding shRNA. Knockdown efficiency was confirmed by real-time PCR quantification (F). Data are means ± SD. ***P < 0.001 compared to the infection control, students’ t test. The cells with indicated gene knockdown were treated with TGF-β1 and then subjected to immunoblotting to evaluate changes in pSMAD1/5/8 and pSMAD2/3 levels (G). Blots are representative of three independent experiments