Fig 2: Muscle histopathology and identification of compound heterozygous LONP1 variants. (A) Histochemical analysis of muscle from LonP1 patient. Serial transverse sections (10 µm) from a muscle biopsy from the patient were subjected to (i) haematoxylin and eosin staining and activity staining for (ii) cytochrome c oxidase (COX) and (iii) succinate dehydrogenase (SDH). (iv) Shows COX: SDH dual staining. Scale bar represents 50 µm. (B) Immunofluorescent analysis of individual muscle fibres from the LonP1 patient. Skeletal muscle sections (20 µm), were subjected to quadruple immunofluorescence as described (13). Signals were measured in individual muscle fibres from antibodies to NDUFB8 (complex I), COX1 (complex IV) and normalized to mitochondrial mass (porin). Each dot represents an individual muscle fibre, colour coded to represent mitochondrial mass (very low = dark purple; low = light purple; normal = yellow; high = orange). Images were used in conjunction with IMARIS software to quantify the levels of COXI and NDUFB8 in individual fibres as described. (C) WES analysis and filtering for rare, autosomal recessive variants in nuclear genes encoding mitochondrial-localized proteins identified only rare heterozygous LONP1 variants.

Fig 3: Inhibition of PLA2G2A induces mitochondrial dysfunction in PDAC cells. (A,B) Effect of PLA2G2A knockdown by siRNA on mitochondrial transmembrane potential. PANC-1 and AsPC-1 cells were transfected with 50 nM siRNA against PLA2G2A for 72 h and then stained with potential-sensitive chemical probe Rhodamine-123 followed by flow cytometry analysis. The green numbers indicate the subpopulation of cells that lost transmembrane potential in the respective samples. (C) Quantitation of cellular ATP in PANC-1 and AsPC-1 cells transfected with 50 nM siRNA against PLA2G2A or with control RNA for 72 h. (D) Western blot analysis of mitochondrial respiratory chain proteins in PANC-1 and AsPC-1 cells transfected with 50 nM siRNA against PLA2G2A or with control RNA for 72 h (representative of 3 independent experiments). NDUFB8, NADH:ubiquinone oxidoreductase subunit B8. (E) PANC-1 and AsPC-1 cells were transfected with 50 nM siRNA against PLA2G2A for 72 h. The cells were then incubated with 5 µM of MitoSOX probe for 30 min. Quantification of mitochondrial superoxide was analyzed by flow cytometry. (F) Quantitation of cellular ATP in PANC-1 and AsPC-1 cells incubated with the indicated concentrations of tanshinone I. (G) PANC-1 and AsPC-1 cells were incubated with 5 µM of tanshinone I for 72 h. Quantification of mitochondrial superoxide was analyzed by flow cytometry after the cells were stained with 5 µM of MitoSOX probe for 30 min. Statistical analyses: data are presented as the mean ± SD of three independent experiments; unpaired t-test for (B,C,E,G); one-way ANOVA for (F). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns = non significant.

Fig 4: Respiratory chain complexes in complementation of ATP synthase deficiency. WB quantification in (A) liver and (B) heart of subunits of the Complex I (CI, NDUFA9, NDUFS3, NDUFB8), III (CIII, CORE2), and IV (CIV, COX4) related to Complex II (CII, SDHA) content was performed in control SHR rats and in SHR-Tmem70ko/ko,tg/0 (tg/0) or SHR-Tmem70ko/ko,tg/tg (tg/tg) rats derived from SHR-Tmem70tg/tg transgenic lines 130 and 126. Data are mean ± SEM, n = 3–6, ** p ≤ 0.01, *** p ≤ 0.001.

Fig 5: Correlation between the High OXPHOS Status and the Abundance of Mitochondrial Complex I, Both at mRNA and Protein Level in the Primary PDAC Cancer CellsHigh OXPHOS patients are depicted in red, and low OXPHOS are in blue.(A) Canonical correlation analysis: graphical representation of PDX using the averaged components 1 and 2 of transcriptomic and metabolic datasets that were retrieved from canonical PLS.(B) Gene set enrichment analysis: the two plots show the enrichment score for the related mitochondrial pathway on the second canonical PLS component in (A).(C) Representative immunoblot showing the abundance of the 5 mitochondrial proteins (ATP5A, UQCR2, SDHB, COXII, and NDUFB8) belonging to mitochondrial complexes (V, III, II, IV, and I, respectively). Time exposure of the blot was 1 min. Ponceau red staining was used as the control of equal protein loading. Mitochondrial complexes protein levels were quantified from western blotting (WB) by ImageJ and normalized to Ponceau red staining. p value from Mann-Whitney test.(D) Representative immunoblot showing the abundance of the mitochondrial protein NDUFB8 belonging to mitochondrial complex I. Time exposure of the blot was 1 min. Complex I protein was quantified by ImageJ and normalized to Ponceau red staining used as control of equal protein loading. p value from Mann-Whitney test.(E) Metabolic analysis by HRMAS-NMR for 6 primary PDAC cancer cells derived from PDX, 3 high OXPHOS (27, 74, and 84) and 3 low OXPHOS (22, 32, and 85). OPLSDA score plot showing the discrimination between high OXPHOS and low OXPHOS.(F) Kaplan-Meier survival curve using transcriptomic analysis on patient-derived xenografts, divided into high and low NDUFB8 gene expression groups (n = 40 and n = 35, respectively; the cutpoint was 13.22). p value was calculated by the log-rank test.See also Figures S5 and S6 and Table S2.