Fig. 1. Effects of BMP4 and Notch signaling on C2C12 differentiation. (A) C2C12 cells were analyzed 6 days post-addition of differentiation medium, and after BMP4 (50 ng/ml) and/or -secretase inhibitor (L-685,458; 1 µM) treatment. Myocyte differentiation was visualized by immunostaining for MHC (red; left panel) and DAPI staining (blue; right panel) was used to identify cell nuclei. Note that a considerable proportion of the cells were MHC-positive under normal differentiation conditions (no addition), and that the addition of BMP4 strongly reduced differentiation (BMP4). Treatment with L-685,458 (L-685,458) led to an increase in the number of differentiated cells compared with treatment with differentiation medium alone, and the combined treatment with BMP4 and L-685,458 (L-685,458 + BMP4) resulted in an increase in the number of MHC-positive cells, as compared with treatment with BMP4 alone. (B) C2C12 cells were analyzed for MHC expression 2 days post-addition of differentiation medium in the presence of BMP4 (50 ng/ml), and after transfection of CSL R218H (upper panel) or ß-gal (lower panel).