Fig. 4

Mitochondrial protein and respiration in iPSC-derived endothelial cells with TOM7 variant. (a) Volcano plot showing mitochondrial proteomic analysis of the differential abundance of proteins in TOM7PL/PL iEndo cells when compared with WT iEndo cells. (b) Functional enrichment analysis of mitochondrial proteome revealed a down or up representation of proteins in GO biological process terms. (c) Immunoblotting of differentially abundant protein components in whole-cell lysate and mitochondria-enriched lysates from iEndo cells. (d) Quantitative analyses of the level of PCK2 in whole cell or mitochondria-enriched lysates from (c). Statistical analysis was performed using Friedman test followed by Dunn's post hoc test, with comparisons made to WT cells. (e) Quantitative analyses of the level of ATP5A in whole cell or mitochondria-enriched lysates. Statistical analysis was performed using Friedman test followed by Dunn's post hoc test, with comparisons made to WT cells. (f) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measured by the Seahorse Mito Stress Assay with 3 × 104 iEndo cells. One representative result was shown. Oligo., oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A. (g) Basal respiration, ATP-coupled respiration, percentage of respiration used for ATP synthesis, maximal mitochondrial respiratory capacity, and proton leak-linked respiration were calculated from one representative experiment (f). Statistical analysis was performed using Kruskal–Wallis test with Dunn's multiple comparisons test. (h) ATP production rates from mitochondrial respiration and glycolysis. Statistical analysis of mitochondrial ATP and glycolytic ATP was performed using the Kruskal–Wallis test followed by Dunn's post hoc test, with comparisons made to WT cells for each parameter. (i) Basal glycolysis and compensatory glycolysis in iEndo cells measured by glycolytic proton efflux rate (PER) in Seahorse assay. Statistical analysis was performed using Kruskal–Wallis test with Dunn's multiple comparisons test. (j) The activities of Complex I (NADH dehydrogenase) and Complex III (cytochrome c reductase) in iEndo cells were measured, with normalization to the input concentration of intact mitochondria. The Y-axis represents the activity levels (mUnits/μg) of Complex I and Complex III at each Δ[(t + 1) −t] 30-s interval. Statistical analysis was performed using Friedman test followed by Dunn's post hoc test, with comparisons made to WT cells. WT, wild-type; Het, heterozygous TOMM7+/PL; Homo, homozygous TOMM7PL/PL; KO, TOMM7 knock-out. Data were presented as mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.