Fig. 3

Genetic inactivation of Notch signaling pathway significantly diminishes BMP9-induced osteogenic differentiation of MSCs. (a) Restoration of PS1 expression in PS1^−/−/PS2^−/− MEFs. The PS1^−/−/PS2^−/− double-knockout (DKO) MEFs were stably transduced with retroviral vector expressing Flag-tagged PS1 (DKO-PS1) or empty vector (DKO-EV). Sunconfluent stable line cells were lysed and subjected to Western blotting analysis using antibodies against the N-terminal fragment of PS1 (PS1-NTF) (a), nicastrin (b) or β-actin (c). Each assay was done in two independent batches of experiments. Representative results are shown. b, c Double-knockout PS1^−/−/PS2^−/− significantly blunts BMP9-induced ALP activity in MSCs. Subconfluent DKO-EV or DKO-PS1 cells were infected with AdBMP9 or AdGFP. ALP activity of the infected cells were determined either by qualitative histochemical staining at day 5 (b) or by quantitative chemiluminescence assay at 3, 5, or 7 days after infection (c, panel a). ALP activity for different titers of AdBMP9 infected DKO-EV or DKO-PS1 cells, was also determined at three days after infection (c, panel b). Each assay condition was done in triplicate. Representative results are shown. **p < 0.001. d Double-knockout PS1^−/−/PS2^−/− significantly inhibits BMP9-induced expression of late osteogenic markers in MSCs. Subconfluent DKO-EV or DKO-PS1 cells were infected with AdBMP9 or AdGFP for five days. Cells were fixed and subjected to immunocytochemical staining with the osteocalcin (Ocn) (a) or osteopontin (Opn) (b) antibody. Minus primary antibody staining was used as a negative control (data not shown). Each staining condition was done in duplicate. Representative results are shown. e Double-knockout PS1^−/−/PS2^−/− significantly inhibits BMP9-induced matrix mineralization in MSCs. Subconfluent DKO-EV or DKO-PS1 cells were infected with AdBMP9 or AdGFP 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