Figure 5
T63 activates BMPs/Smad1/5/8 pathway. (a) Expression of Bmp2, Bmp4 and Bmp7 genes. MC3T3-E1 cells were treated with T63 (5 μM) for 48 h before qRT-PCR analysis (n = 3). *p < 0.05, **p < 0.01 versus control. (b) Activation of p-Smad1/5/8 in both dose- and time-dependent manner in the presence or absence of BMP2. The cells were treated with T63 (5 μM) as indicated (upper panels), or with the indicated concentration for 0.5 h (lower panels) in the presence or absence of BMP2 (50 ng/ml), followed by western blot analysis. Relative optical density for each band was quantified, normalized and labeled under each lane. (c) Noggin decreased RUNX2 protein level. MC3T3-E1 cells were treated with T63 (5 μM) in the presence or absence of Noggin (200 ng/ml) for 48 h, and subjected to western blot analysis (n = 3). (d) Noggin decreased T63-induced ALPL activity. MC3T3-E1 cells were treated with Noggin (200 ng/ml) in the presence of T63 (10 μM) for 6 days and the ALPL activity was measured. **p < 0.01 versus control, # p < 0.05 between both groups (n = 3). (e) Noggin decreased T63-induced mineralization. MC3T3-E1 cells were treated with the indicated compounds for 18 days before subjected to Alizarin Red S staining, and the quantification of calcified nodules were plotted as means ± SD (n = 3). *p < 0.05 versus control, # p < 0.05 between both groups. Full-length blots are shown in Supplementary Fig. S5.
