FIG. 8.

TNF-α inhibits C2C12 myogenesis and stabilizes cyclin D1. C2C12 cells were induced to differentiate in the absence or presence of 20 ng of TNF-α per ml. Cytokine addition was repeated at 6 h and every additional 12 h after the induction of differentiation. At 72 h, the cells were washed with PBS, fixed for 10 min at room temperature with 4% paraformaldehyde, and photographed by phase-contrast microscopy. In parallel treatment cultures, cells were harvested at 24 and 48 h and whole-cell lysates were prepared for Western blot analysis. A 50-μg portion of total protein was fractionated by SDS-polyacrylamide gel electrophoresis, and immunoblotting was performed to probe for cyclin D1. The blot was subsequently stripped and reprobed for cdk4, used as an internal control.