Extended Data Fig. 3: ABHD18 regulates mitochondrial respiratory chain assembly and generates MLCL from nCL.

(a) BN-PAGE immunoblots (total OxPHOS, CII, CIII, CIV, CV) and in-gel activity assays (CI, CIV) from isolated mitochondria of HAP1 mutant cell lines. Blots probed for individual complexes and supercomplexes. Total OXPHOS (n = 5 biological replicates) obtained using antibody cocktail targeting CI, CII, CIII, CIV, and CV. CIII and CIV blots (n = 3 biological replicates), and CIV activity are representative (n = 2 biological replicates). (b) Bar plots of Seahorse Cell Mito Stress Test for respiratory parameters: basal respiration, maximal respiration, mitochondrial ATP generation, and glycolytic ATP generation across mutant HAP1 cell lines. Data presented are means ± s.d. of n = 4 technical replicates. Scatterplots of OCR and ECAR measurements taken during seahorse assays (right). (c) Dot plots of lipidomics analysis of CL and MLCL abundance in HAP1 WT and mutant cell lines (left and middle panel respectively). Relative abundance shown as bar plot above dot plot for each genotype. Bar plot quantifying MLCL/CL ratio changes across HAP1 cell lines using total CL and MLCL species abundance (right). (d) Dot plots showing lipidomics analysis of CL and MLCL abundance in an additional set of HAP1 mutant cell lines (n = 3 clonal mutant cell lines, n = 4 WT cell lines). (e) Thin Layer Chromatography (TLC) showing wild-type ABHD18[WT], but not catalytic dead ABHD18[S199A] recombinant protein deacylates different cardiolipin species to yield MLCL and DLCL, and deacylates MLCL to yield DLCL. Negative control does not contain any ABHD18 protein present, only buffer.