Fig. 4

1 mM Pyruvate and 200 μM uridine supplementation rescues the metabolic consequences of OXPHOS dysfunction in fibroblasts. A Schematic representation of the central carbon metabolic reactions using NAD+/NADH as cofactors depicting the treatment mechanisms of pyruvate + uridine. Thickness of the arrows and the size of the metabolites indicate the magnitude of alteration in OXPHOS deficient fibroblasts compared to control. B Effect of pyruvate + uridine supplementation on the doubling time, based on 72-hours IncucyteⓇ proliferation experiments (Controls n = 8; OXPHOS deficient n = 11; PDH deficient n = 2; technical replicates >3 per cell line) with calculation of doubling time over a 48-hour period starting 24 h after plating. C Tracer metabolomics faith of glucose, glutamine and pyruvate in 1 mM pyruvate and 200 μM uridine supplemented cultures of controls, OXPHOS and PDH deficient fibroblasts with U–¹³C glucose, U–¹³C glutamine or U–¹³C pyruvate. A simplified map of glycolysis and TCA cycle is depicted. The area of the circles represents the relative abundance of the metabolite compared to average control. Pink, dark blue, light blue and grey colour represent the fractional labelling coming from glucose, glutamine, pyruvate or other sources, respectively. D The relative change in abundances of aspartate, E malate and F glycerol-3-phosphate in controls, OXPHOS deficient and PDH deficient fibroblasts upon supplementation with 1 mM pyruvate and 200 μM uridine. G Fractional carbon labelling of citrate in control, OXPHOS deficient and PDH deficient fibroblasts cell lines supplemented with 1 mM pyruvate and 200 μM uridine, cultured with U–¹³C glucose, U–¹³C glutamine or U–¹³C pyruvate. H Comparison of the 6 isotopologues of citrate in control, OXPHOS deficient and PDH deficient fibroblasts cell lines cultured with U–¹³C glucose or I U–¹³C glutamine after supplementation with 1 mM pyruvate and 200 μM uridine. Tracer metabolomics in control (n = 8), OXPHOS deficient (n = 9) and PDH deficient (n = 2) fibroblast cell lines (technical replicates 1–3 per cell line). Abundances normalized for protein content (BCA) and to the average of the controls per experiment. Statistics: one-way ANOVA with post-hoc Dunnett's T3 multiple comparison tests, effect of treatment was assessed using paired student T-tests comparing no treatment with a treatment and the error bars are +/−SD. αKG: alpha-ketoglutarate; dpf: days post fertilisation; Glycerol-3-P: Glycerol-3-phosphate; OXPHOS: oxidative phosphorylation system; PDH: pyruvate dehydrogenase; P + U: 1 mM pyruvate +200 μM uridine.