FIG. 2.

The rictor-mTOR complex is sensitive to rapamycin, and differentiation of C2C12 cells is inhibited by depletion of rictor but not raptor. (A) C2C12 myoblasts were maintained in GM or DM with or without rapamycin (100 ng/ml) or deprived of four amino acids (−4aa; Leu, Lys, Met, and Gln) for 3 days. Cells were fractionated as described in Materials and Methods, and proteins were immunoprecipitated (IP) from the membrane, cytosolic, and nuclear fractions with an anti-mTOR antibody. The coimmunoprecipitated proteins were detected by anti-rictor and anti-raptor antibodies. Normal rabbit IgG was used as a control. (B) C2C12 myoblasts were left uninfected (lanes C2) or infected with a lentivirus encoding a control shRNA (lanes S) or rictor shRNA (lanes E5). After 24 h, the medium was replaced with DM. C2C12 myoblasts without rapamycin (lanes C2) or with rapamycin (lanes R+; 100 ng/ml) or lentivirus-infected cells were harvested after incubation in DM for 1 to 4 days. Cell lysates were prepared, and proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted as described in Materials and Methods. (C) C2C12 myoblasts were transfected with siRNA against raptor (R5, smart pool) or a control siRNA (lanes S) or mock transfected (lanes C2 and R+). After 24 h, the medium was replaced with DM. C2C12 myoblasts without rapamycin (lanes C2), with rapamycin (lanes R+), with control siRNA (lanes S), or with raptor siRNA (lanes R5) were harvested after incubation in DM for 1 to 4 days. Cell lysates were prepared, and proteins were separated by SDS-PAGE and immunoblotted as described in Materials and Methods. (D) C2C12 cells were infected with lentiviruses with rictor shRNA (E5 or E6, two different plasmids for packaging), control shRNA, or lentivirus encoding GFP for 24 h, the medium was replaced with DM, and the cells were incubated for 5 days. Cells were photographed, and representative microscope fields are shown. (E) Parental C2C12 cells or C2C12 cells infected with lentiviruses encoding rictor shRNA or control shRNA or transfected with raptor siRNA were incubated in DM in the absence (C2C12) or presence of rapamycin 100 ng/ml (Rap+) for 1 to 4 days. MyHC was detected by immunofluorescence. (F) The percentages of the total cells that were MyHC positive (black bars) and multinucleated (gray bars) were calculated by counting 20 random microscope fields for each condition.