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Fig. 5. Transient transfection with pMyoD increases ALP activity in C3H10T1/2 cells. (A) C3H10T1/2 cells were transiently transfected with MyoD expression vector (pEMSV-MyoD/PGK-puro) and empty vector (PGK-puro) and treated for 0, 1, 3 and 6 days with or without BMP-7 (400 ng/ml). ALP activity in the cell lysate was measured using p-nitrophenylphosphate as a substrate. An increase in BMP-7 induced ALP activity in MyoD transfected cultures above that induced in the control cultures, was evident after 3 days and was further increased at 6 days post-transfection. (B) Representative cultures of transfected cells were double-stained for ß-Gal (green) and for ALP activity (red) after a 3-day incubation in differentiation media with or without BMP-7 (400 ng/ml). ß-Gal staining was used to identify the transfected cells independently of any response to BMP-7 treatment. Double-stained cells are evident in the cultures transfected with MyoD and treated with BMP-7. Scale bar, 80 µm.

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