FIGURE 9.

Inhibition of mouse C2C12 myoblast differentiation by FBI-1 can be rescued by transfection with recombinant adenovirus overexpressing Rb gene. 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, 2, and 4 after treatment with 2% horse serum differentiation medium. Differentiation into myotubes was observed only in control cells at day 4. Stable C2C12 myoblast cells overexpressing FBI-1 were differentiation into myotubes only when transfected with recombinant adenovirus overexpressing Rb gene at day 4, but not in the cells rescued with control adenovirus. Ad, recombinant adenovirus. B, Western blot analysis of the C2C12 control or C2C12-FBI-1 cell lysates using the antibodies indicated. Rb expression was 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. Transfection with recombinant adenovirus increases Rb expression both in control and stable cells. Cell extracts (40 μg) were analyzed by Western blot analysis. GAPDH, control; Rb, retinoblastoma protein.