Figure 1

MG132 inhibited TNFα-induced proteasome activation in differentiated C2C12 cells. (A) Western blot showing IκBα expression in differentiated C2C12 pre-treated with vehicle (DMSO) or MG132 for 1 h followed by stimulation with TNFα (50 ng/ml) for 5, 10, 15, 30, and 120 min in presence or absence of MG132. GAPDH was used as a loading control. (B) Graphical representation of IκBα expression levels over time indicating that a pre-treatment with MG132 (dashed line) was able to prevent IκBα degradation compared to cells pre-treated with the vehicle (DMSO) (solid line). (C) C2C12 myoblasts co-transfected with the NFκB-luciferase and Renilla luciferase reporter vectors. They were then differentiated and stimulated with TNFα for 6 h in the presence or absence of MG132. The ratio of Luciferase:Renilla activities indicates that the presence of MG132 prevented the binding of NFκB to the luciferase promoter. (D) Graphs showing MuRF-1 and Atrogin/MAFbx qPCR expression data from C2C12 myotubes stimulated or not with TNFα (50 ng/ml) for 1 h. TNFα caused the expression of the muscle-specific ubiquitin ligases MuRF-1 and Atrogin/MAFBx. (E) To evaluate the impact of MG132 on MuRF-1 and Atrogin/MAFbx expression, differentiated C2C12 were pre-treated or not with MG132 (40 μM) for 1 h before being stimulated with TNFα (50 ng/ml) for 1 h. Expression of MuRF-1 and atrogin/MAFbx was quantified by qPCR. MG132 inhibited the expression of MuRF-1 and atrogin/MAFbx. Data are presented as the means ± SEM of at least four independent experiments performed in triplicate. (*p < 0.05, **p < 0.005, ***p < 0.001 compared to control).