Figure 6

Disruption of calcium homeostasis (but not caspase signaling) is a primary mechanism by which C2-Ceramide induces cytotoxicity in dopaminergic MN9D cells. A, The MTS assay for cell viability in diff-MN9D cells. Co-treatment with the pan-caspase inhibitor Z-VAD (25 μM) or the caspase-8-specific inhibitor Z-IETD (25 μM) attenuated cytotoxicity in diff-MN9D cells induced by C2-Cer concentrations at and below 5 μM but not above 5 μM. All values represent group means +/− SEM, n = 3–4. One-way ANOVA with Tukey’s post-hoc test to compare the effect of specified C2-Cer concentrations on diff-MN9D viability to vehicle without caspase inhibitors in the MTS assay, where ### denotes p < 0.001 compared to vehicle or between two C2-Cer concentrations. Two-way ANOVA with Tukey’s post-hoc to compare effects of caspase inhibitors at each C2-Cer concentration where * denotes p < 0.05 and *** denotes p < 0.001 compared to no caspase inhibitor. B, Pre-loading of diff-MN9D cells with cell permeant BAPTA-AM (10 μM) to buffer intracellular Ca²⁺ transients significantly attenuated cytotoxicity induced by C2-Cer. Cell viability was assayed by MTS reduction. One-way ANOVA with Tukey’s post-hoc test; *** denotes p < 0.001 compared to no BAPTA at each concentration.