C18-ceramide-LC3B-II interaction on mitochondrial membranes is regulated downstream of Drp1-mediated mitochondrial fission. (a-b) Effects of Gly120Ala and Phe52Ala conversions, which perturbed C18-ceramide-binding of LC3B, on targeting mitochondria by LC3B-containing autophagosomes (visualized by confocal microscopy using anti-FLAG and anti-Tom-20 antibodies; a) or mitochondrial function (measurement of oxygen consumption OCR; b). (c) Roles of siRNA-mediated knockdown of p62, NIX and Drp1 in the alteration of OCR in the absence or presence of CerS1/C18-ceramide induction (-/+Tet) were measured using the SeaHorse. Data shown are an average of at least three experiments ± s.d. (*P <0.05). (d) Effects of siRNA-mediated knockdown of Drp1 on the localization of ceramide within mitochondrial membranes were visualized using the co-localization of anti-ceramide (red) and anti-Tom20 (green) antibodies under confocal microscopy, in the absence or presence of CerS1/C18-ceramide induction (-/+ Tet) compared to Scr-siRNA-transfected controls (right and left, upper and lower panels, respectively). Scale bars represent 10 microns in (a) and (d).