FIGURE 2.

Rheb, but not S6K1, negatively regulates myoblast differentiation. C2C12 myoblasts were transduced overnight with lentiviruses expressing shRNAs for Rheb (A–C) or S6K1 (D–F) (Scramble or Scr as a non-targeting control), and subjected to puromycin selection for 2 days followed by differentiation for 3 days. A and D, differentiated myocytes were stained for MHC (green) and DAPI (red). B and E, myocytes were quantified for differentiation index, fusion index, and average myotube size (myonuclei number per myotube). C and F, cells were lysed for Western analyses, and band intensities were quantified by densitometry and normalized to tubulin control. All data shown are mean ± S.D. (n = 3 for each condition). For B & E, paired t test was performed to compare each data to Scramble control. For C and F, one-sample t test was performed to compare each data to Scramble control. *, p < 0.05; **, p < 0.01.