Fig 2: Effect of CoC3 on mitochondrial protein expression levels in control and mutant fibroblasts. Western blot analysis of the affected mutant protein in each cell line: mtND3 (A), NDUFAF6 (B), COX15 (C), NDUFS1 (D), COQ7 (E), NDUFS4 (F). VDAC was used as mitochondrial mass marker, mtND3 and mtCO2 as mitochondrial-encoded reference proteins and NDUFA9 and nCOX4 as nuclear-encoded mitochondrial reference proteins. Overall, all patients show alterations with respect to the control, which are partially reversed after CoC3 treatment. “C” stands for healthy control cells. CoC3 treatment: 1 µM Pterostilbene, 5 µM nicotinamide, 1 µM riboflavin, 1 µM thiamine, 1 µM biotin, 5 µM lipoic acid and 1 µM L-carnitine. Differences between patients are probably due to the high variability in mutation severity and their genetic background. A representative actin band is shown, although loading control was additionally checked for every Western blot. Band densitometry can be found in Supplementary Figure S5.

Fig 3: Effect of CoC3 on complex I and complex IV activities in control and mitochondrial mutant fibroblasts. Complex I proteins’ activities were measured using Complex I Enzyme Activity Dipstick Assay Kit from Abcam, with the exception of mutant COX15. In this particular mutation complex IV was measured. Mitochondrial complex activity in mtND3 cells (A), NDUFAF6 cells (B), COX15 cells (C), NDUFS1 cells (D), COQ7 cells (E), NDUFS4 cells (F). Results are shown in the dipstick images both in colour (bluish for complex I and yellowish for complex IV) and black/white. Band intensity was quantified using the Image Lab software. “C” stands for healthy control cells. Fibroblasts were treated for 7 days with CoC3 treatment: 1 µM Pterostilbene, 5 µM nicotinamide, 1 µM riboflavin, 1 µM thiamine, 1 µM biotin, 5 µM lipoic acid and 1 µM l-carnitine. * = p < 0.01 between Control and mutant fibroblasts. a p < 0.01 between untreated and treated mutant fibroblasts. a.u. (Arbitrary unit).

Fig 4: Effect of CoC3 on the expression levels of UPRmt-related proteins in control and mitochondrial mutant cell lines. Western blot analysis of several UPRmt -related proteins: Eif2a and P-eif2a proteins as Integrated Stress Response (ISR) markers; ATF4, ATF5 and CHOP proteins as canonical UPRmt proteins; HSP60 and mtHSP70 proteins as chaperones; SIRT3 protein as main target of pterostilbene; Nrf2 protein as a modulator of mitochondrial antioxidant pathways; NAMPT enzyme as NAD+/NADH cell metabolism regulator. “C” stands for healthy control cells. Fibroblasts were treated for 7 days with CoC3 treatment: 1 µM Pterostilbene, 5 µM nicotinamide, 1 µM riboflavin, 1 µM thiamine, 1 µM biotin, 5 µM lipoic acid and 1 µM L-carnitine. Mutations in figure panels: mtND3 (A), NDUFAF6 (B), COX15 (C), NDUFS1 (D), COQ7 (E), NDUFS4 (F). A representative actin band is shown for all assays, although loading control was checked for every Western blot. Band densitometry is shown in Supplementary Figure S7.

Fig 5: Screening of compounds in galactose medium. Cells were initially seeded in DMEM high glucose. After 3 days, glucose medium was changed to galactose. Images were acquired right after changing the medium and after 72 h of incubation. In optimal conditions control and both mutant cell lines present a similar proliferation rate (A–C). Control cells almost do not alter their growth rate (D) but mutant ND3 and NDUFAF6 cells are unable to survive (E and F). Pterostilbene (1 µM) treatment does not affect control cells (J) but promotes the survival of mutant ND3 and NDUFAF6 cells in galactose medium (H and I). In addition, cocktail CoC3 (1 µM Pterostilbene, 5 µM nicotinamide, 1 µM riboflavin, 1 µM thiamine, 1 µM biotin, 5 µM lipoic acid and 1 µM L-carnitine) significantly improves pterostilbene’s positive effects (J–L). CoC3 without ptersotilbene was also examined, but no favorable effect could be observed on mutant fibroblast (N and O), suggesting that pterostilbene is necessary for the positive effect of CoC3. The quantification of cellular proliferation is shown in Supplementary Figure S4. Scale bar = 40 µm.