Fig. 6. Stable transfection of MyoD enhances osteogenic response of C3H10T1/2 cells to BMP. (A) Isolated clones of C3H10T1/2 cells, stably transfected with either MyoD-expression vector (10T-MD) or its empty vector (10T-puro), were cultured for 3 days in differentiation media with or without BMP-7 (400 ng/ml) and then double-stained for MyoD expression (black/brown) and ALP activity (blue). MyoD was only detected in the 10TMD lines incubated without BMP. BMP-treatment decreases MyoD expression and increases ALP-positive cells. More ALP-positive cells are evident in 10TMD cell lines than in the 10Tpuro cell lines (arrows). Scale bar, 80 µm. (B) RT-PCR analyses of selected genes in clonal cell lines incubated for 3 days with or without BMP-7 (400 ng/ml). BMP-treatment decreased the expression of MyoD and AchR in 10TMD-12 and increased the expression of ALP, consistent with the inhibition of myogenesis and the stimulation of osteogenesis. Higher ALP activity (A, BMP) is reflected in the increased ALP mRNA in the MyoD-transfected culture incubated with BMP-7.