Fig. 4. A specific role for caspases during TNFα-mediated inhibition of myogenic differentiation. (A) C2 cells cultured in DM suplemented with or without TNFα and caspase inhibitors, as indicated. Immunolocalization of myosin was used as a marker of differentiation. Quantitative analysis of myogenic differentiation (% differentiation) was performed as described in Materials and methods. Pan-caspase inhibitors BAF or z-VAD are able to rescue differentiation of C2 cells in the presence of TNFα, while the caspase-3 inhibitor DEVD is ineffective. In F3 cells, which are insensitive to TNFα, all the caspase inhibitors have no major effect upon differentiation. (B) Caspase activity (shaded bars) was measured in TNFα-treated C2 cells and expressed as fold increase versus controls (untreated C2 cells). To rule out cross-reactivity of the substrates with caspase-3, parallel experiments were carried out by incubating the cell cultures with DEVD-FMK before performing the caspase activity assay (solid bars). Only caspase activities that are significantly induced by TNFα (caspase-1, -5, -6, -8 and -9 in C2 cells) are shown. Significance was calculated using a one-sample t-test (p <0.05).