Fig. 1
BMP9-induced osteogenic differentiation of MSCs is inhibited by the γ-secretase inhibitor Compound E. (a) Most components of Notch signaling pathway express at a low level in MSCs. Total RNA was isolated from subconfluent C3H10T1/2 (a) and iMEFs (b) cells and subjected to semi-quantitative PCR (a) or TqPCR (b) analysis of the expression of Notch receptors and ligands. (b) The γ-secretase inhibitor Compound E (CE) inhibits BMP9-induced osteogenic marker ALP activity in MSCs. Subconfluent C3H10T1/2 cells were infected with AdBMP9 or AdGFP and treated with the indicated concentrations of CE. At 5 days of treatment, the cells were fixed and subjected to ALP histochemical staining (a). Alternatively, the cells were lysed at the indicated time points and subjected to chemiluminescence–based quantitative assay of ALP activity (b). Each assay condition was done in triplicate. *p < 0.05, **p < 0.001. (c) BMP9-induced matrix mineralization is effectively blunted by CE. Subconfluent C3H10T1/2 cells were infected with AdBMP9 or AdGFP, treated with the indicated concentrations of CE, and maintained in the mineralization medium for 14 days. The cells were fixed and subjected to Alizarin Red S staining. Each assay condition was done in triplicate. Representative results are shown
