FIG. 9.

Effect of ectopic expression of cyclin D3 on MCK expression in growing and differentiating C2 myoblasts. Proliferating C2 myoblasts were transfected with 0.5 μg of the MCK luc reporter plasmid in the presence of the indicated amount of the cyclin D3 expression construct (Rc/CMV-cycD3); the Rc/CMV expression vehicle without an insert was included to normalize DNA in all transfections. Eighteen hours after transfection, the cells were trypsinized; one half of the cells were plated onto 90-mm-diameter dishes, the other half were plated onto 60-mm-diameter dishes, and refed with growth medium. Parallel transfections were directly transferred to differentiation medium. After 72 h the cells in 90-mm-diameter dishes were subconfluent (A), those in 60-mm-diameter dishes were confluent (B), and those in differentiation medium were fully differentiated (C); at that time cells were collected and assayed for luciferase and β-galactosidase activities. Equal amounts of proteins from each cell lysate were also assayed for cyclin D3 expression by Western blotting. The results shown are from one representative experiment. MCK-luc activity is expressed relative to the levels detected in the absence of cyclin D3. The experiments were done in duplicate and repeated three times. The mean values (expressed as fold induction relative to the baseline values) and the standard deviations (S.D.) of these experiments are shown at the bottom.