Figure 3.

Induction of endogenous C₁₈-ceramide by CerS1 expression mediates lethal mitophagy. (a) Effects of induction of wt-CerS1 versus its catalytically inactive mutant, which cannot generate C₁₈-ceramide, expression (containing V5 tags) on the formation of LC3B-II were determined by western blot (first panel, +/-tet, respectively). Successful induction of wt- and mutant-CerS1 expression was confirmed using the anti-V5 antibody. Beta-actin was used as a loading control. Full blots can be found in Supplementary Figure 13. (b) Generation of endogenous C₁₈- and C_18:1-ceramides in response to wt-CerS1 compared to the mutant-CerS1 induction was measured by LC/MS/MS. (c) Effects of wt-CerS1/C₁₈-ceramide induction (+tet) on GFP or LC3B-GFP lipidation were visualized using confocal microscopy compared to non-induced controls (-tet). Scale bars represent 10 microns. (d) Roles of wt-CerS1/C₁₈-ceramide versus the catalytically inactive mutant-CerS1 induction (+tet) in the regulation of mitochondrial function was assessed by measuring oxygen consumption rate using the SeaHorse, compared to non-induced controls (-tet). (e) Targeting mitochondria with autophagolysosomes in the absence or presence of wt-CerS1/C₁₈-ceramide (-/+ tet, repectively) was visualized by co-localization of MTG and LTR using confocal microscopy. (f) Effects of wt and mutant CerS1 (-/+ tet) on ATP generation. Scale bars represent 10 microns. (g) Effects of wt-CerS1 (-/+ tet) on ATP generation in the absence or presence of shRNA-mediated knockdown of LC3B were measured. Data shown are an average of at least three experiments ± s.d. (*P <0.05).