Figure 5

TNF- and ceramide-induced caspase-3 cleavage was attenuated by SMase inhibitors and caspase inhibitors rescued differentiated MN9D cells from TNF-induced cytotoxicity. A, Diff-MN9D cells were treated for 3 days with 5 ng/mL TNF in the presence or absence of the ASMase inhibitor Desipramine (Des, 5 μM) or the NSMase inhibitor GW4869 (10 μM) and were thereafter harvested for SDS-PAGE and immunoblot analysis of total caspase 3 or cleaved caspase 3. B, Quantification of western blot analysis of caspase 3 and cleaved caspase 3. One-Way ANOVA with Tukey’s post-hoc test to compare inhibitor conditions to TNF alone, where ** denotes p < 0.01, ***denotes p < 0.001. C, MTS assay for cell viability in diff-MN9D cells. TNF induced dose-dependent death of diff-MN9D cells and was caspase-dependent. Co-treatment with TNF plus the pan-caspase inhibitor Z-VAD (25 μM) or with TNF plus the caspase-8-specific inhibitor Z-IETD (25 μM) robustly blocked TNF-induced cell death in diff-MN9D cells. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test to compare the effect of specified TNF concentrations on diff-MN9D viability without caspase inhibitors in the MTS assay, where # denotes p < 0.05, ## denotes p < 0.01, and ### denotes p < 0.001 compared to ‘Veh’ or between two concentrations. Two-way ANOVA with Tukey’s post-hoc to compare effects of caspase inhibitors at each TNF concentration where *** denotes p < 0.001 compared to no caspase inhibitor.