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.

Reversine increases cellular plasticity of 3T3E1 osteoblasts and hSMs. (A) The 3T3E1 osteoblasts (plated at 6,000 cells per cm² in GM) were cultured in the presence of 300 nM reversine (RE) for 4 days. The compound was then removed, and cells were grown in AI conditions for an additional 6 days. (B) hSMs cells (plated at 3,000 cells per cm² in GM) were cultured in the presence of 300 nM reversine for 4 days. The compound was then removed, and cells were grown in OI or AI conditions (OIC or AIC) for an additional 9 days. The cells were analyzed by histocytochemistry and RT-PCR. DMSO was used as a negative control in all experiments. Red, ALP, Oil red O; blue, HT.

Affinity chromatography and target identification. (A) Structures of positive (AM·reversine+) and negative (AM·reversine−) affinity matrices. (B) Silver staining and Western blot analysis of proteins retained by affinity supports. (C and D) Reversine inhibits the enzymatic activity of active MEK1 in a dose-dependent fashion as indicated by ERK2-phosphorylation (C) and reversine inhibits the ATPase activity of myosin II (heavy chain) in vitro (D). (E) Reversine (20 nM) blocks MEK-dependent ERK1/2-phosphorylation in C2C12 cells. C2C12 cells were treated with 20 nM reversine for 1 h, and cells were lysed and analyzed by Western blot. DMSO was used as a negative control. U0126 (10 μM) was used as a positive control. (F) Overexpression of MEK1 or NMMII can partially block reversine's activity. C2C12 myoblasts (plated at 6,000 cells per cm² in GM) transiently transfected with MEK1 or NMMII (with FuGENE6) were cultured in the presence of 20 nM reversine for 48 h. The compound was then removed, and cells were cultured in OI or AI conditions for an additional 6 days and analyzed by histocytochemistry. Empty vector was used as a negative control (Mock). (G) C2C12 myoblasts transfected with siNMMIIs (with X-treme) and cultured in the presence of 10 μM U0126 in GM for 48 h gain multipotency. C2C12 myoblasts (plated at 6,000 cells per cm² in GM) transiently transfected with siNMMIIs were cultured in the presence of 10 μM U0126 for 48 h. The compound was then removed, and the cells were cultured in OI or AI conditions for an additional 6 days and analyzed by histocytochemistry. Cells transfected with NTsi and treated with DMSO were used as a negative control. Red, ALP, Oil red O; blue, HT.

Cell cycle effects. (A) Treatment with reversine induces G₂/M phase accumulation in C2C12 myoblasts. C2C12 myoblasts treated with 20 nM reversine in GM for 48 h were analyzed by FACS based on DNA content (by using PI staining). (B) C2C12 myoblasts were cultured in GM in the presence of 20 nM reversine for 48 h. After removal of reversine (0 h), cells cultured in GM were analyzed by FACS. (C) C2C12 myoblasts synchronized at G₂/M phase and treated with 10 μM U0126 gain multipotency. C2C12 myoblasts synchronized at G₁/S, S, or G₂/M phase were analyzed by FACS. After release from synchronization by the addition of fresh GM in the presence or absence of 10 μM U0126, cells were incubated for 4 h. The compound was then removed, and the cells were cultured in OI or AI conditions for an additional 6 days and analyzed by histocytochemistry. Red, ALP, Oil red O; blue, HT.