Fig 2: Tissue-specific expression and compensation of the ANT family.(A) Western blots of isolated mitochondria from various mouse tissues for ANT1, ANT2, ANT4, and a loading control for electron transport chain complex V (ComV). ANT2 protein is up-regulated in all five tissues analyzed to compensate for the loss of the Ant1 gene. ANT2 is up-regulated in testis in response to loss of the Ant4 gene. These results are representative of three independent experiments. (B) Western blots for ANT2 and ANT4 from isolated liver mitochondria from the indicated gene-targeted mice showing that ANT4 is induced for the first time in the absence of the Ant1 and Ant2 genes. A loading control for ComV is shown. These results are representative of three independent experiments. (C) Quantification of ANT4 expression normalized to ComV expression, as in (B) (n = 3; *P = 0.05). (D) Western blots of ANT1, ANT2, ANT4, and a loading control for ComV, performed using extracts of isolated liver mitochondria from Ant1-/- Ant4-/- Ant2fl/fl (ANT2-only) and Ant1-/- Ant4-/- Ant2fl/fl-Alb-Cre (Ant-triple-null) mice. Data are representative of three independent experiments. (E) Quantification of ANT2 expression normalized to ComV expression, as in (D) (n = 4; *P = 0.05). (F) Representative fluorescence-activated cell sorting analysis of TMRE-stained mouse liver mitochondria isolated from Ant1-/- Ant4-/- Ant2fl/fl versus Ant1-/- Ant4-/- Ant2fl/fl-Alb-Cre mice. The TMRE fluorescence threshold is shown in the bottom of the panel versus when no fluorescence signal is detected above background. FCCP is a potent uncoupler of mitochondrial respiration, which is provided to depolarize mitochondria. Results are representative of three independent experiments.

Fig 3: The genomic and transcriptomic variations of DEMs in colon cancer.A DEMs of SLC25 with differential DNA mutation rates and mRNA expression levels in colon cancer from the TCGA dataset were identified using cBioPortal. Among them, SLC25A4, SLC25A5, SLC25A24, SLC25A34, SLC25A3, SLC25A51, SLC25A44, SLC25A12, SLC25A32, and SLC25A15 exhibited higher mutation frequencies, with rates of 5, 6, 6, 10, 11, 11, 12, 16, 21, and 25%, respectively. B No statistically significant differences in OS and disease-free survival (DFS) were observed in patients with colon cancer grouped by DNA mutation rate in the altered and unaltered groups by log-rank test, despite a longer overall survival time in the unaltered group with a lower DNA mutation frequency in DMEs of SLC25. C An UpSet diagram exhibited alternative splicing for DMEs and suggested that exon skipping was the major type of alternative splicing event. D The locations of copy number variation in DMEs of 23 SLC25 human chromosome pairs. E Patients with SLC25A5 overexpression showed a significantly high sensitivity to the ROCK inhibitor GSK269962A and to foretinib. Upregulation of SLC25A24 increased tansapimycin, trametinib, and refametinib resistance.

Fig 4: Expression of mitochondrial SLC25 in colon cancer.A Heatmap showing the expression of 53 SLC25 members between colon cancer and normal colon tissues according to the TCGA-COAD dataset. Thirty-seven of 53 were differentially expressed members (DEMs). B Spearman’s rank correlation analyses between 37 DEMs at the transcriptional level. Negative and positive correlations are marked blue and red, respectively. The scatter plot shows the correlation between SLC25A10 and SLC25A39 expression as an illustrative example. C Most of the 37 DEMs interacted at the protein level. The color of the nodes represents the differential expression level, while the size of the nodes indicates the degree of importance in the network. D Ten of 37 DEMs and stage I or II diseases were relevant in this analysis by Wilcoxon rank-sum test, showing that patients with earlier tumor stage tended to have higher mRNA expression of SLC25. E Kaplan–Meier curves suggested that patients with upregulated SLC25A5 and SLC24A24 had a longer overall survival (OS) time.

Fig 5: Characterization of HeLa-?g cells.a Western blot analysis of indicated protein in total cell lysates of HeLa-?g cells (40 µg/lane). Images are representative of three independent blots. b Western blot analysis of isolated mitochondria from wild-type (WT) and ?g cells for the indicated F-ATP synthase subunits. Histogram refers to the quantification of protein levels relative to citrate synthase (CS) and represents mean ± SEM of 3 (for subunit a) or 4 (for all other subunits) independent blots, *p < 0.05, two-sided Student’s t-test. Gray bars, WT and cyan bars, HeLa-?g cells. c Clear native-PAGE analysis and subsequent immunoblotting against ATP synthase subunits ß, c, and against SDHA of WT and ?g mitochondria in the presence of indicated amount of digitonin (g digitonin/g protein). Images are representative of two independent blots. d Western blot on isolated mitochondria for ANT2 and ANT3. Histogram refers to the quantification of protein levels relative to citrate synthase (CS) and represents mean ± SEM of three independent blots. Two-sided Student’s t-test. e Mitochondrial oxygen consumption rate (OCR) was evaluated in intact cells by Seahorse XF Analyzer before and after the addition of oligomycin (O), FCCP (100 nM), rotenone (R), and antimycin A (AA). Traces are average of four independent experiments for WT (gray trace) and ?g cells (cyan trace). Basal and oligomycin-sensitive OCR is expressed as mean ± SEM of four independent experiments, ***p < 0.001 with two-sided Student’s t-test. f Swelling assay in isolated mitochondria in the presence (black traces) or absence (red traces) of Ca2+. PTP opening was induced with 50 µM Ca2+, and alamethicin (ala) was added where indicated. Histograms refer to the fraction of swollen mitochondria after about 9 min of Ca2+ addition and are mean ± SEM of six independent experiments, ***p < 0.001, two-sided Student’s t-test.