FIGURE 8.

FBI-1 inhibits mouse C2C12 myoblast differentiation by inhibiting Rb gene expression. A, differentiation of control mouse C2C12 myoblasts and stable C2C12 myoblasts overexpressing FLAG-FBI-1, by horse serum treatment. Pictures of the cells were taken at days 0–4 after treatment with 2% horse serum differentiation medium. Differentiation into myotubes was observed only in control cells at days 3 and 4. Stable C2C12 myoblast cells overexpressing FBI-1 were prepared by transfection with either control lentri virus or recombinant Lenti-M1.4-FLAG-FBI-1 virus. B, Western blot analysis of the C2C12 cell lysates using the antibodies indicated. Rb expression was gradually increased in the control cells with concomitant decrease in FBI-1 (LRF). LRF is a mouse homologue of human FBI-1. In the stable cells, although endogenous LRF is decreased, FLAG FBI-1 is stably expressed and repressed Rb expression. Cell extracts (40 μg) were analyzed by Western blot analysis. GAPDH, control; Rb, retinoblastoma protein. C, myogenic differentiation is inhibited by FBI-1 in the stable C2C12-FLAG-FBI-1 cells. However, control cells underwent myogenic differentiation and showed multinuclear myotubes at days 3–4.