Figure 1.

Cyclin D3 knockdown in myoblasts leads to precocious onset of differentiation. C2.7 myoblasts transduced either with the retrovirus expressing cyclin D3-specific shRNA (shCyclin D3) or with the empty retrovirus (control) were seeded at 2 × 10⁵ cells into 90-mm dishes, and transferred to DM after 48 hours. Total RNA and whole-cell extracts were prepared from cells collected in GM 24 hours after seeding or at indicated times after exposure to DM. (A): RT-qPCR analysis was performed on the genes indicated. Graphs represent average 2^−ΔCT values ± SEM from four independent experiments. (B): Western blot analysis was performed using antibodies specific for the indicated proteins. (C): Quantification of protein expression by densitometry analysis of Western blots shown in (B); values are presented as fold change relative to control myoblasts cultured in GM (set to 1), after normalization to the corresponding values of α-tubulin levels. Asterisks denote significance (*, p < .05; **, p < .01; ***, p < .001). Abbreviations: DM, differentiation medium; GM, growth medium; MHC, myosin heavy chain; Rb, retinoblastoma gene; pRb, retinoblastoma protein.