Fig. 6: GRAMD1C regulates mitochondrial bioenergetics.

a Wt and GRAMD1C knockout (GKO) cells expressing 3XHA-EGFP-OMP25 were subjected to mitochondria isolation from a total cell lysate (TCL). The isolated mitochondria were incubated with the cholesterol probe mCherry-D4 (recombinant protein shown in lane 7) in the presence or absence of methyl-β cyclodextrin (MBCD). The asterisk indicates free mCherry. b Quantification of mCherry-D4 band intensity in the isolated mitochondria fractions from a. relative to the average band intensity of the mitochondrial proteins TOM20 and COXIV and normalized to Wt cells. Significance was determined using two-tailed Students T test from n = 3 independent experiments. Error bar = SEM. P value = 0.0238. c Mitochondria from Wt or GKO cells were isolated and mitochondrial lipids were extracted. Absorbance changes corresponding to cholesterol abundance were normalized to mitochondrial protein concentration. Significance was determined using Student’s T test from n = 3 independent experiments. Error bar = SEM. P value = 0.0162. d Cells treated with control siRNA (siNC) or siGRAMD1C were treated to high-pressure freezing followed by freeze substitution (1% OsO4, 0.5% uranyl acetate, 0.25% glutaraldehyde in acetone). Arrows with m point towards mitochondria. Scale bar = 5 µm, inset scale bar = 1 µm. n = 31 cells. e Mitochondrial oxygen consumption rate (OCR) was analyzed in control, and GRAMD1C knocked down cells using the Seahorse analyzer. OCR was measured after the gradual addition of Oligomycin (oligo), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and Rotenone/Antimycin A (Rot/AntiA). f ATP-linked respiration is calculated from the difference between the maximal respiratory capacity and the proton leak. Significance was determined using one-way ANOVA followed by Bonferroni’s comparison test from n = 3 experiments. Error bar = SEM. P value = 0.0294 and 0.0413. g OCR was analyzed in Wt, GKO, GKO + GRAMD1C, and GKO + ΔGRAM cells using the Seahorse analyzer. h ATP-linked respiration is calculated from the difference between the maximal respiratory capacity and the proton leak. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test from n = 3 experiments. Error bar = SEM. P value = 0.0159. Source data are provided as a Source Data file.