Extended Data Fig. 5: ABD646 treatment mimics ABHD18 LOF phenotypes.

(a) Dot plots showing lipidomic analysis of CL and MLCL abundance in HAP1 and TAZ-c1 KO cells treated with either DMSO or 1μM of ABD646 for 5 days (left and middle panel respectively). Bar plot quantifying MLCL/CL ratio changes across HAP1 cell lines using total CL and MLCL species abundance (right). (b) Bar plots illustrating the proliferative effect of 10 μm ABD646 on HAP1 and TAZ KO cells. All values normalized to HAP1 DMSO (n = 3 biological replicates), with significance determined by one-way ANOVA followed by Dunnett’s post-hoc test. (c) TEM images of HAP1 TAZ KO cells treated with DMSO or ABD646 for five days. Green: mitochondria. Scale bars, 1 μm (top) and 200 nm (bottom). (d) Schematic illustrating mode of action of three substrate oxidation pathway inhibitors for glucose/pyruvate (UK5099), long chain fatty acids (LCFA, Etomoxir), and glutamine oxidation (BPTES). (e) Bar plots of seahorse substrate oxidation tests using Etomoxir, BPTES, and UK5099 on HAP1 WT and mutant cells, with ABD646 or DMSO treatment. Data are maximal respiration values (OCR pmol/min, n = 6 technical replicates). (f) Representative TEM images showing partial restoration of mitochondrial morphologies observed in BTHS fibroblasts treated with 1μM of ABD646 for 5 days. Green: mitochondria. Scale bars: 1 μm, and 200 nm. n = 2 biological replicate cell pellets per treatment. (g) Flow cytometry analysis of mitochondrial mass (Mitotracker Green FM) and membrane potential (Mitotracker CMXROS) in fibroblasts from patients with BTHS treated with vehicle or 5μM ABD646 for five days. (h) BN-PAGE immunoblots and in-gel activity assays for mitochondrial complexes in fibroblasts from patients with BTHS treated with vehicle or 5μM ABD646 for five days. (i) Dot plots showing lipidomics analysis of CL and MLCL abundance in BTHS fibroblasts treated with either DMSO or 1μM of ABD646 for 5 days (left and middle panel respectively). Bar plot quantifying MLCL/CL ratio changes across HAP1 cell lines using total CL and MLCL species abundance (right). (j) Representative images at day 5 of zebrafish embryos injected with increasing concentrations of TAZ MOs (2.5ng-10.0 ng) (left; scale bars, 200 μm). Bar plot quantifying observed phenotypes across doses of TAZ MOs (right). (k) 24 h acute treatment of ABD646 has chronic benefit for TAZ MO zebrafish. Representative images of zebrafish embryos following either a 24-h treatment with ABD646 on WT (n = 45), and TAZ MO treated (n = 40) embryos. Embryos were washed once a day for 5 consecutive days with imaging and quantification on day 5 (left; scale bars, 200 μm). Right, quantification of embryos demonstrating “rescue” or near WT phenotypes per wash day. (l) Box plot of ABHD18 gene expression across HAP1 and TAZ mutant clones in nutrient rich (IMDM) or nutrient limiting (DMEM) media conditions. Data are median read counts ± interquartile range (IQR), n = 3 biological replicates per media condition. Significance determined by one-way ANOVA followed by Dunnett’s post hoc test. (m) Bar plot of spearman correlations between TAZ and ABHD18 gene expression across tissue samples. Data from the Genotype-Tissue Expression (GTEx) Project.