Figure 2. Notch inhibition ablates BMP-induced chondrocytes maturation.

Primary mouse sternal chondrocytes were cultured in regular media and treated with DAPT (5 uM) and/or BMP2 (100 ng/ml) before being harvested for AP staining, total RNA and protein isolation at the indicated time points. (A) BMP treatment resulted in significant increases in AP staining at days 3, 5, and 7, compared to vehicle-treated controls. (B) Real time PCR showed a markedly decreased expression of Hes1, AP, MMP13, Runx2, p57 in chondrocyte after treatment with DAPT for 7 days. (C) Western blot analysis of p-SMAD1/5/8 expression in chondrocytes after 6 hours of DAPT and/or BMP treatment. β-actin was used as a loading control. (D) Quantization of band density of western blot results showed the folds over control. (E) Immunofluorescence data showed an increased expression of p-SMAD1/5/8 in nuclear area in BMP2 treated cells and a decreased labeling in both peri-nuclear and nuclear areas in DAPT treated cells. PCR data are means ± SD of three independent experiments performed in duplicate and the control gene expression level was set at 1. (*p < 0.05 compared with control at same time point; ^#p < 0.05 compared with DAPT alone cells).