Extended Data Fig. 5. HcMel12 and 4434 mutant cells recapitulate the cellular phenotypes observed in B78-D14 cells.

a Heteroplasmy changes upon subsequent transfection of melanoma cell lines (n = 3–6 biological replicates). b Immunoblot of indicative respiratory chain subunits. Representative result of three biological replicates is shown. c mtDNA copy number (n = 9 technical replicates over n = 3 biological replicates). d Basal oxygen consumption rate (OCR) (n = 36, 21, 12 and 12 technical replicates over n = 12, 7, 4 and 4 biological replicates) e Proliferation rate of cell lines in permissive growth media (n = 12 and 6 technical replicates over n = 4 and 6 biological replicates) f Energy (adenylate) charge state (n = 24, 25, 18 and 18 technical replicates over n = 9, 9, 6 and 6 biological replicates). g NAD+:NADH ratio (n = 24, 25, 17 and 18 technical replicates over n = 9, 9, 6 and 6 biological replicates). h GSH:GSSG ratio (n = 22, 21, 15 and 16 technical replicates over n = 9, 9, 6 and 6 biological replicates). i Heatmap of unlabelled steady-state abundance of select metabolites in Hcmel12 cells. Succ. cys, succinylcysteine. (n = 24–25 technical replicates over n = 9 biological replicates). j Heatmap of unlabelled steady-state abundance of select metabolites in 4434 cells. Succ. cys, succinylcysteine. (n = 15–18 technical replicates over n = 6 biological replicates). Heatmap of k U-¹³C-glucose- and L U-¹³C-glutamine-derived select metabolites in Hcmel12 and 4434 cells (n = 6–18 separate technical replicates over n = 2–6 biological replicates). Volcano plot showing detected differences in protein abundance of m B78-D14 wild-type versus B78 mt.12436^80% cells N Hcmel12 wild-type versus Hcmel12 mt.12436^83% cells and o 4434 wild-type versus 4434 mt.12436^72% cells (n = 3 biological replicates). Heatmaps of protein abundances for p complex I, q complex II, r complex III, S complex IV and t complex V nuclear and mtDNA-encoded subunits in B78 mt.12436^80%, Hcmel12 mt.12436^83% and 4434 mt.12436^72% cells compared to their respective wild-type (n = 3 biological replicates). u Venn diagram comparing proteomic changes of mutant versus wild-type per cell line. v Significant KEGG pathway changes of mutant versus wild-type per cell. A student’s one-tailed t-test (C-H, I-L), student’s two-tailed t-test (P-T), two-tailed Wilcoxon signed rank test (M-O) or Fishers exact test (FDR = 1%) (V) were applied. Error bars indicate SD. Measure of centrality is mean. * P = < 0.05, ** P = <0.01, *** P = <0.001, **** P = <0.0001. Number of replicates are described across conditions from left to right as presented. Heatmap representations of data where asterisks are not present report non-significant changes.

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