Fig 2: Overexpression of UQCRH in HCC. (A) UQCRH mRNA expression in HCC (T) and corresponding adjacent liver tissues (N), determined with semiquantitative RT-PCR. NL indicates normal liver. (B) Box plot analysis illustrating differences in UQCRH mRNA expression levels between normal (n = 10), adjacent liver (n = 30), and HCC (n = 96) tissues. UQCRH expression was measured using real-time RT-PCR, with 18S rRNA as an internal control. (C) UQCRH protein level in pair-marched HCC (T) and corresponding adjacent liver tissues (N) was determined by western blotting. *, More than 3/2 times of upper quartile.

Fig 3: Histology of the heart (haematoxylin and eosin staining and electron microscopy) Representative photomicrographs of heart tissue, stained with haematoxylin and eosin, from wild-type (WT, upper panel) and Uqcrh -/- (lower panel) mice at 9 weeks of age. Normal histopathological structure of the myocardium (red box, central panels) and aorta (black box, right panels) were observed in both groups of mice. Scale bars represent 1 mm in the left panels and 50 µm in the central and right panels.Electron microscopy images of 3 representative wild-type (WT, upper panel) and 3 Uqcrh -/- mice (lower panel) heart mitochondria. Mitochondria from Uqcrh-/- mouse heart tissue showed paracrystalline inclusions (arrows). Magnification 1:50,000.

Fig 4: Two-exon deletion in UQCRH alters supercomplex stoichiometries and stabilityMitochondrial membranes from fibroblasts of control (WT) and patient fibroblasts (i, immortalised) (upper panels) and heart tissue from wild-type (WT) and Uqcrh -/- mice (lower panels) were solubilised with digitonin and separated on native gradient gels. BN-PAGE gel stained with Coomassie showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I) = immortalised fibroblasts).BN-PAGE gel stained with NADH:NTB reductase activity stain showing molecular weight marker (BHM) and samples from control and patient (primary fibroblasts, (I) = immortalised fibroblasts).Further complexome analysis of the BN-PAGE gels shown in A and B. Each native lane was cut into even fractions from high to low molecular mass. Proteins in each slice were digested with trypsin and analysed by quantitative mass spectrometry. Shown here are average IBAQ values of all identified subunits from complexes III, I and IV (full data including all individual subunits shown in Fig EV4).Profile of complex III (averaged IBAQ values) in the patient (dashed line) and control (solid line) fibroblasts.Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in control (solid lines) and patient (dashed lines) fibroblasts.BN-PAGE gel stained with Coomassie (left) and NADH:NTB reductase activity stain (right) showing molecular weight marker (BHM) and heart tissue samples from wild-type (WT) and Uqcrh -/- mice.BN-PAGE and immunoblot using the antibody to CIII subunit UQCRC2 in heart tissue samples from wild-type (WT) and Uqcrh -/- mice.Further complexome analysis of the BN-PAGE gels shown in F, shown here are average IBAQ values of subunits from complexes III, I and IV (full data including all individual subunits shown in Fig EV4).Profile of complex III (averaged IBAQ values) in wild-type (+/+, solid line) and Uqcrh -/- (-/-, dashed line) heart tissue.Profile of complexes III (red), I (yellow) and IV (green) to visualise and assess supercomplex composition and stability in wild-type (+/+, solid line) and Uqcrh -/- (-/-, dashed line) heart tissue.

Fig 5: Biochemical assessment of patient fibroblasts and Uqcrh -/- mice shows impaired complex III activity and stability Respiratory chain enzyme activities from human control (green) and patient (red) fibroblasts. Mean activities of controls (n = 8) are set to 100% and error bars represent 1 standard deviation. Data are normalised to citrate synthase (CS) activity.Respiratory chain enzyme activities in heart, brain and liver, of wild-type and Uqcrh -/- mice. Data are normalised to citrate synthase (CS) activity. Values are given as mean ± SD, n = 4 WT, 5 Uqcrh -/- (heart), 3 WT, 3 Uqcrh -/- (brain), 1 WT and 2 Uqcrh -/- (Liver). *P < 0.05, ****P < 0.0001, 2-way ANOVA (multiple comparisons). Data showing CS activity/total protein concentration can be found in Appendix Fig S1A.Western blot analysis of OXPHOS components in the patient (P) and control (n = 2, C1-2) fibroblasts.BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from patient and control fibroblasts (n = 2, C1-2) solubilised with DDM. Immunoblotting was performed using antibodies to a subunit of each complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1] and CV [ATP5A]).Western blot analysis of liver lysates derived from 6 wild-type (WT) and 6 Uqcrh -/- animals at 4 weeks of age. The upper band (97 kDa) refers to loading control VCP protein; the lower band (11 kDa) indicates UQCRH.Immunohistochemical staining of UQCRC2 was performed on liver, heart and kidney derived from wild-type and Uqcrh -/- mice at 9 weeks of age. Staining of VDAC1/porin was also carried out (Appendix Fig S1B).Graphs indicate score values of immunohistochemical staining intensity of both VDAC1/porin and UQCRC2. Data are given as mean ± SEM. *P < 0.05; n = 3, Student’s t-test (unpaired samples). Source data are available online for this figure.