Fig. 2. (A) Myogenic specification factors are suppressed in G0 and activated in G1. Western blot analysis of total protein shows that both MyoD and Myf5 are present in asynchronous adherent cultures of C2C12 myoblasts (Mb) but not in C3H fibroblasts (Fb) as expected. 72 hour myotube cultures (Mt) strongly express MyoD but not Myf5. Neither MRF is expressed after 12 or 48 hours of suspension culture (S12, S48). Replating for 2 to 30 hours (R2-R30) causes induction of MyoD earlier (at R6) than Myf5 (at R18). Data are representative of three independent experiments. (B) Synchronization in G0 does not induce differentiation. Northern blot analysis of RNA isolated from asynchronous, arrested, reactivated or differentiated C2C12 cells. The differentiation marker MCK is only detected in myotubes (Mt), but not in asynchronous myoblasts (Mb), at 12 or 48 hours in suspension (S12, S48) or at 2-30 hours after replating (R2-R30). Histone mRNA indicates the extent of DNA synthesis. MyoD and Myf5 mRNAs correlate with the protein data in Fig. 2A. Data are representative of three independent experiments.