Fig. 4.

NAADP signaling is essential for differentiation of C2C12 cells. C2C12 cells were induced to differentiate in the presence of DMSO, 200 nM bafilomycin, or 100 μM Ned-19. (A) RNA was harvested at the indicated time points and analyzed for expression of myogenin and skNAC by Northern blot analysis. (B) Following 4 d of differentiation, cells were stained for myosin heavy chain and DAPI to determine the differentiation index (percent nuclei in myosin heavy chain-positive cells). (C) Bar chart (mean with SEM; n = 4) representing the differentiation index following treatment for 4 d with control, bafilomycin, or Ned-19. Recovery of C2C12 differentiation following removal of Ned-19 was demonstrated by (D) Northern blot, (E) myosin heavy chain and DAPI staining, and (F) calculation of the differentiation index.