Fig. 2
Dominant-negative mutant Notch1 inhibits BMP9-induced osteogenic differentiation of MSCs. a Schematic representation of the dominant-negative Notch1 (dnNotch1), which contains the extracellular ___domain and the transmembrane region of mouse Notch1. b Adenovirus-mediated overexpression of dnNocth1. Subconfluent C3H10T1/2 cells were infected with AdR-dnNotch1 or AdRFP control virus for 36 h. Total RNA was collected and subjected to semi-quantitative PCR (sqPCR) using primers for mouse Notch1 extracellular ___domain or mouse Gapdh. The sqPCR was done in triplicate; and representative results are shown. c, d dnNotch1 inhibits BMP9-induced ALP activity in MSCs. Subconfluent C3H10T1/2 cells were co-infected with AdBMP9 or AdGFP and different titers of AdR-dnNotch1. At five days after infection, the ALP activity of the infected cells were determined either by qualitative histochemical staining (a) or quantitative chemiluminescence assay (b). Each assay condition was done in triplicate. Representative results are shown. *p < 0.05, **p < 0.001. e dnNotch1 diminishes BMP9-induced matrix mineralization. Subconfluent C3H10T1/2 cells were co-infected with AdBMP9 or AdGFP and/or AdRdnNotch1 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
