Extended Data Fig. 1: Characterising complex I truncating mutations in melanoma.
a Unique truncating variants were detected in an average of 16% of melanoma patients (n = 281) and in 19% of melanoma patients (n = 89) from the Hartwig Medical Foundation (HMF) and MSK IMPACT melanoma cohorts respectively. b Truncating and c non-truncating mutations in melanoma patients, combined from both IMPACT and HMF patient cohorts, show that truncations are enriched in complex I, compared to complexes III, IV, and V. d Immunoblot of DdCBE pair expression post-sort. αHA and αFLAG show expression of left (TALE-L) and right TALEs (TALE-R) respectively. One biological replicate performed. e Heteroplasmy measurements of cells generated (n = 6 biological replicates). f Off-target C>T activity of DdCBEs on mtDNA. Figure shows mutations detected at heteroplasmies >2% and is a measure of mutations detected relative to wild-type. These mutations likely do not impact our key observations as both models behave similarly across experiments. g Immunoblot of indicative respiratory chain subunits. Representative result of three biological replicates is shown. Volcano plot showing detected differences in protein abundance of wild-type versus h mt.1243660% cells and i mt.1194460% cells. Differences of p-value < 0.05 and log2 fold change > 0.5 shown in red (n = 3 biological replicates). Heatmaps of protein abundances for j complex I, k complex II, l complex III, m complex IV and n complex V nuclear and mtDNA-encoded subunits (n = 3 biological replicates). o mtDNA copy number (n = 22 and 21 technical replicates over n = 8 and 7 biological replicates). p Expression of mitochondrial genes (n = 4 biological replicates). q Basal oxygen consumption rate (OCR) (n = 18 and 12 technical replicates over n = 6 and 4 biological replicates) r Energy (adenylate) charge state (n = 9 technical replicates over n = 3 biological replicates). s Measurements of the electrical component of the proton motive force, ΔΨ, the chemical component of the proton motive force ΔpH and total protonmotive force, ΔP (n = 4 biological replicates). t NAD+:NADH ratio (n = 9 technical replicates over n = 3 biological replicates). u GSH:GSSG ratio (n = 6, 10, 12, 8 and 10 technical replicates over n = 2, 4, 4 and 3 biological replicates). A high GSH:GSSG ratio represents a more reductive intracellular environment. v GSH:GSSG ratio (n = 9 technical replicates over n = 3 biological replicates) w Mitochondrial NADH oxidation state (n = 4 biological replicates). P-values were determined using a two-tailed Fisher’s exact test (A-C), student’s one-tailed t-test (E, O, Q, R, T, V), two-tailed Wilcoxon signed rank test (H, I), one-way ANOVA test with Sidak multiple comparisons test (J-N) or one-way ANOVA test with Fisher’s LSD test (S, U, W). Error bars indicate CI (A-C) or SD (E,O-W). 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.
