Fig 2: BMP2, TGF-β1 and BMP8A show different effects on the expressional induction of Nog, Grem1 and Grem2. C3H10T1/2 cells were treated with BMP2 (1 nM), TGF-β1 (0.3 nM), BMP2 plus with TGF-β1 or BMP8A (1 nM) for 24 h. The transcript levels (upper panel) and encoding protein amounts (lower panel) of Nog (A), Grem1 (B) and Grem2 (C) were then evaluated. β-actin amounts served as loading controls in immunoblotting. D To explore how BMP8A induces Grem1 expression, cells were treated with BMP8A in the absence or presence of SB431542 (SB) or dorsomorphin (DM) as indicated. Quantification data are presented as means ± SEM. *P < 0.05, ***P < 0.001 compared to no-treatment control. # P < 0.001 compared between indicated groups

Fig 3: BMP8 activates both SMAD pathways in bone marrow-derived mesenchymal stem cells. A–C Detection and autocrine function of BMP8 proteins in bone cells. A Conditioned media collected from bone marrow (BM) cells or C3H10T1/2 cells without or with Bmp8a knockdown were subjected to immunoblotting using the antibody against BMP8. B Knockdown efficiency of Bmp8a in C3H10T1/2. ***P < 0.001. C Knockdown of Bmp8a dampens the phosphorylation levels of both SMAD1/5/8 and SMAD2/3 endogenously. To confirm that the BMP8-induced SMAD signaling occurs in MSCs, primary human BM-MSCs (D), primary mouse BM-MSCs (E) or C3H10T1/2 cells (F) were treated with graded doses of BMP8A, BMP2, activin A or TGF-β1 as indicated. The cell lysates were then subjected to immunoblotting using antibody against phosphorylated SMAD1/5/8 (upper panel) or phosphorylated SMAD2/3 (lower panel). Total forms of SMADs and β-actin served as loading controls

Fig 4: Selected TGF-β superfamily ligands show different effects on the regulation of antagonist expression. C3H10T1/2 cells were treated with 1 nM BMP2 (A), 0.3 nM TGF-β1 (B) or 1 nM BMP8A (C) for 24 h. The transcripts of all TGF-β superfamily antagonists were then quantified. All data were expressed as fold changes by normalizing against the own transcripts of each gene in cells without ligand treatments. Data are shown as means ± SEM. n = 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001