Fig 2: DIDS reduces cisplatin-induced impaired mitochondrial respiration chain in L-02 hepatocytes. (A) Mitochondrial respiratory chain COXI activity was detected. *P < 0.05 versus the NC group and # P < 0.05 versus the DDP group. ANOVA with LSD. Error bars indicate SD, n = 6. (B) Western blotting analysis of COXI subunit NDUFB6 protein in L-02 hepatocytes. (C) Quantification of NDUFB6 relative protein expression. ANOVA with LSD. Error bars indicate SD, n = 3.

Fig 3: TMEM126A is associated with a stable CI subassembly of the ND4-module(A) Scatterplots generated from the liquid chromatography-mass spectrometry (LC-MS) analysis of CI immunopurified fractions performed on the same mitochondrial-enriched fractions used for the proteomic analyses in Figure 3. The values of the logarithmic fold change (log2 H/L) for each protein in experiment 1 (exp.1; light untreated, heavy Dox treated) are represented on the x axis. The logarithmic fold change values (-log2 H/L) derived from experiment 2 (exp.2; heavy untreated, light Dox treated) are represented on the y axis. Each point represents a specific protein. CI subunits and assembly factors are highlighted; CI subunits of the same module are the same color. “Uncertain” subunits are those with still unclear assignment. TMEM126A is shown in red. Proteins with statistically significant changes are also labeled.(B) Immunodetection of TMEM126A and NDUFS3 on western blots of whole-cell lysates and mitochondrial enriched fractions from 143B-/- NDUFS3 cells treated with 100 ng/mL Dox for 0 (untreated), 1, 2, 4, and 8 days (n = 2). TMEM126A band signal intensities were quantified by densitometry and normalized to the signal of either vinculin for whole-cell lysates, and CS or voltage-dependent anion channel (VDAC) for mitochondrial fractions. The mean values of the treated cells are the percentage of those of the untreated control. Data are means ± SD.(C) Immunodetection of TMEM126A and NDUFB6 (ND4-module) in western blots of enriched-mitochondrial fractions from 143B-/-NDUFS3 cells treated with 100 ng/mL Dox for 0 (untreated), 1, 2, 4, and 8 days, solubilized using digitonin and separated by BN-PAGE. Two asterisks (**) indicate a subassembly containing TMEM126A and co-migrating with NDUFB6 subassembly; one asterisk (*) indicates a lower-molecular-sized subassembly with positive staining for TMEM126A.The two panels correspond to the same samples separated in the same BN-PAGE gel and transferred onto the same membrane, which was subsequently cut in two for immunodetection. SDHA (CII) was used as loading control in BN-PAGE blots. VDAC was used as loading control in western blots of the same samples separated by SDS-PAGE.(D) Immunodetection of TMEM126A, NDUFB6 (ND4-module), UQCRC2 (CIII), and MT-CO2 (CIV) on western blots of mitochondrial enriched fractions from 143B-/-NDUFS3 cells treated with 100 ng/mL Dox for 0 (untreated) and 8 days, solubilized with 4 mg digitonin/mg protein and resolved by 2D BN-PAGE. All the subunits were immunodetected on the same blot. One asterisk (*) indicates a signal derived from the anti-TMEM126A antibody with which the membrane was previously incubated.

Fig 4: Two-dimensional electrophoretic analysis of different COX forms present in SURF1-/- mouse tissues and fibroblasts and SURF1 patient fibroblasts.Respiratory complexes and supercomplexes were solubilized using 8 g digitonin/g protein of isolated mitochondria, separated by BNE in the first dimension and SDS PAGE in the second dimension and detected by Western blots using specific antibodies to COX1 (cIV), CORE1 (cIII), NDUFB6 (cI) and NDUFS3 (cI). For analysis, heart (A), muscle (B), liver (C), brain (D) and fibroblast (E) of SURF1+/+ and SURF1-/- mice as well as fibroblasts (F) of human control and SURF1 patient were used. COX1 assembly intermediates (COX1 AI), COX monomer (M), COX dimer (D), III2–IV SC (III2–IV), I–III2 SC (I–III2), I–III2–IVn SCs (I–III2–IVn), complex III dimer (cIII2), complex I (cI).

Fig 5: In vivo spermatocytes have heterogeneous mitochondria and reduced OXPHOS activity.(A) Immunolabeling of NDUFB6, MTCOI, and MPC1 in spermatocytes in testis sections. Line scans are shown as arbitrary units (au) to the right. Scale bars, 20 µm. (B) Quantification showing the percentage of spermatocytes with reduced or heterogeneous staining. L/Z, leptotene/zygotene; P/D, pachytene/diplotene. N = 4. (C) COX/SDH enzyme histochemistry in adult testis sections. Note that mutant sections have much reduced OXPHOS activity. Scale bars, 50 µm. ST, spermatid; SC, spermatocyte; SG, spermatogonium. Data are represented as mean ± SEM. ****p=0.0001; ***p=0.001; **p=0.01. For statistical tests used, see the Materials and methods section.