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Fig. 2. Myosin inhibitors reversibly arrest myoblast proliferation. (A) Dose response of DNA synthesis in asynchronous myoblasts in low serum. Asynchronous myoblasts were treated for 24 hours with medium containing 2% HS alone (C), or 2% HS + vehicle (D) or BDM at 3, 10 or 30 mM (B3, B10, B30 respectively) or ML7 at 1.5, 5 or 15 µM (M1.5, M5, M15, respectively). Cells were pulsed with BrdU and labeled nuclei detected by immunostaining. Both BDM and ML7 elicited a dose-dependent decrease in the frequency of S phase cells (mean ± s.e.m. n=2). (B) Dose response of DNA synthesis in asynchronous myoblasts in high serum. Asynchronous myoblasts were treated with MI in high serum (20% FBS) and DNA synthesis measured as described in A. Whereas BDM effectively inhibited DNA synthesis, ML7's action was not evident in the presence of serum (mean ± s.e.m. n=2). (C) MIs prevent progression to S phase. Myoblasts were synchronized in G₀ by suspension culture, replated in either 2% HS (black bars) or 20% FBS (gray bars) for 24 hours, and DNA synthesis measured as described in Fig. 2A. Vehicle-treated cells (D) entered S phase in a serum-dependent manner. BDM (but not ML7) inhibited S phase entry in a dose-dependent manner in 20% FBS. (mean ± s.d. n=4). (D) Growth arrest by MI is rapid. The kinetics of arrest by MI were measured by treating asynchronous myoblasts for 2-30 hours with 2% HS + DMSO (D), 30 mM BDM (B) or 15 µM ML7 (M). Inhibition of DNA synthesis was evident within 6 hours of MI addition. (mean ± s.e.m. n=2). (E) Arrest is reversible. Myoblasts were arrested by 24 hours treatment (0 hours after drug removal) in 15 µM ML7 or 30 mM BDM compared to vehicle alone. Cumulative BrdU-labeling of cells entering S from 5-28 hours after addition of growth medium showed that 90% were labeled by 22-28 hours. MI-treated cells took 4-8 hours longer to achieve 90% labeling. (F) MI treatment arrests cells in G₀. Asynchronous myoblasts were treated for 24 hours with vehicle, 30 mM BDM or 15 µM ML7 and DNA content measured by FACS. Whereas control cells were distributed in all phases of the cell cycle, 90% of MI-treated cells possessed a G₁ DNA content consistent with arrest in G₀. (mean ± s.e.m. n=2).

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