Reversine (RE) increases the cellular plasticity of C2C12 myoblasts. (A) Chemical structure of reversine. (B) Reversine blocks the terminal differentiation of C2C12 myoblasts. C2C12 myoblasts (plated at 6,000 cells per cm2) were cultured in GM in the presence or absence of 20 nM reversine for 4 days and then stained with hematoxylin (HT). (C) C2C12 myoblasts gain multipotency after 48-h treatment with 20 nM reversine. C2C12 myoblasts (plated at 6,000 cells per cm2) were cultured in GM supplemented with 20 nM reversine for 48 h. After removal of compound, cells were cultured in OI or AI conditions (OIC or AIC) for an additional 6 days and analyzed by histocytochemistry and RT-PCR. (D) Reversine does not induce C2C12 myoblasts to directly transdifferentiate into osteoblasts or adipocytes without LSICs. After 48-h treatment with 20 nM reversine, cells were fixed and analyzed by histocytochemistry. (E) Clonal analysis. Reversine increases the cellular plasticity of C2C12 myoblasts at the single-cell level. C2C12 myoblasts (plated at 1 cell per well in 96-well plate with GM) were grown in the presence of 20 nM reversine for 2 weeks. The resulting colonies were split into two and cultured with OI or AI conditions for an additional 6 days. Plasticity, cells with increased plasticity that can differentiate into adipocytes and osteoblasts; Cell Death, the colonies that underwent apoptosis during the expanding process. DMSO was used as a negative control in all experiments. Red, ALP, Oil red O; blue, HT.