Fig. 1. (A) DNA synthesis in reversibly arrested C2C12 myoblasts. Immunodetection of BrdU in asynchronous myoblasts (Mb), cells synchronized by 48 hours in suspension (S48), activated cells 28 hours after replating (R28) and 3 day myotube cultures (Mt). Arrowheads in R28 point to a labeled mitosis (telophase). In differentiated cultures (Mt), residual cycling myoblasts incorporate label, whereas myotube nuclei do not. (B) All arrested myoblasts progress to S phase upon reactivation. Cumulative DNA synthesis in synchronized C2C12 myoblasts after labeling with BrdU for 2-48 hours of reactivation (R2-R48) shows that 98% of arrested cells re-enter the cell cycle. The extended G1 phase is consistent with inclusion of a G0-G1 transition phase. For comparison, note that asynchronous cells (Mb) labeled for 2 hours show high levels of DNA synthesis, whereas suspension-arrested cells (S) show <2% S phase cells despite labeling for 12 hours. Data represent the means±s.e.m. of duplicate samples per time point. Similar results were obtained with two independent experiments. (C) Synchronous activation of G0 myoblasts requires both adhesion and mitogens. FACS analysis of adherent C2C12 myoblasts (Mb) reveals a DNA content profile typical of an asynchronous population. 48 hours after suspension (S48), most cells show a G1 DNA content consistent with arrest in G0. Replating for 6 hours in GM (R6) or for 24 hours in DM (R24D) does not alter the profile, whereas by 24 hours in GM, adhesion- and mitogen-dependent signals synergize to return arrested cells to S phase (R24G). Data represent the means±s.d. of four independent experiments.