(A) The C2C12 (a,b) and MM14 (c,d) cells were cultured (1.5×105 cells/cm2) under myogenic conditions (a, c) or treated with BMP2 (300 ng/ml) (b,d) for 7 d. Cultures were processed for alkaline phosphatase activity to identify osteogenic lineage (Materials and Methods). In the absence of BMP2, numerous multinucleated myotubes were visible (a,c, arrows) but these cells were negative for enzyme activity. In the presence of BMP2 strong alkaline phosphatase activity alkaline phosphatase (b,d). (B) Cells were cultured for 7 days as above with or without BMP2 and processed for RT-PCR using primers specific for GAPDH, osteocalcin, Runx2, desmin, and MyoD. BMP2 produced a decrease in the myogenic desmin and MyoD expression but induced osteogenic markers osteocalcin and Runx2.