Figure 3.

(A) Inhibition of myoblast differentiation by the PI 3-kinase-specific inhibitor LY294002. CEM were cultured in MG medium for 1 day after seeding, and continued to grow for 3 days in MD medium containing the indicated concentrations of LY294002 or dimethyl sulfoxide solvent. Cells were stained on day 4 with the LeukoStat kit (Fisher) to distinguish the cytoplasm and the nucleus, and representative fields were photographed (×16 objective, brightfield illumination). (B) Inhibition of multinucleated myotube formation by LY294002. Cells on day 4 were stained as above, and counted under ×16 objective lens to determine the percentage of nuclei in myotubes (containing at least three nuclei per cell). Three independent plates with three random fields per plate were used. (C) Inhibition of CK activity by LY294002. CK activity on day 4 was determined in two experiments with three replicate plates in the presence of LY294002 as indicated. (D) Inhibition of muscle-specific gene expression by LY294002. Total protein was extracted from CEM treated for 3 days with LY294002 as indicated and processed for immunoblot assay as described in Fig. 2B.