Figure 6(A-C) Statistical analysis of Seahorse basal OCR and maximal respiration of (A) wt, (B) gas-1(fc21) and (C) gas-1(fc21); cest-2.2 O/E nematodes fed with empty vector (C RNAi), nhr-49 or nhr-80 RNAi (ordinary one-way ANOVA with Dunnett's multiple comparisons test, n=4-12 biological replicates from 3 independent experiments). Data information: bars represent mean ±SEM Across experiments, p value summary is ns=not statistical, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D) Scheme of oxaloacetate biosynthesis. MDH-2 stimulates oxaloacetate biosynthesis, which can be used to support tricarboxylic acid cycle (TCA) and/or cytosolic phosphoenolpyruvate generation by PCK-1. In physiological settings, phosphoenolpyruvate can be used to generate pyruvate, which can be reduced to lactate or oxidized to acetyl-CoA (dashed lines). (E) Lifespan assay of nematodes exposed to control or mdh-2 RNAi bacteria. Data information: the average median lifespan ± SEM across replicates is reported underneath the graphs, and additional information (e.g., n numbers) Lifespans come from the same experiment, although they are represented separately for the sake of clarity Representative Seahorse experiment (F) of untreated- and 24 h oxaloacetate-treated adult gas-1(fc21) nematodes (Unpaired t-test, n=9-10 biological replicates from 3 independent experiments). Data information: points represent mean±SEM. Across experiments, p value summary is ns=not statistical, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Representative maximal respiration (G) of untreated- and 24 h oxaloacetate-treated adult gas-1(fc21) nematodes (Unpaired t-test, n=9-10 biological replicates from 3 independent experiments). Data information: bars represent mean ±SEM Across experiments, p value summary is ns=not statistical, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (H) Representative lifespan analysis of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals exposed to control and pck-1 dsRNA. Data information: the average median lifespan ± SEM across replicates is reported underneath the graphs, and additional information (e.g., n numbers) Lifespans come from the same experiment, although they are represented separately for the sake of clarity (I-J) Representative OCR of 5-day-old gas-1(fc21); cest-2.2 O/E nematodes grown on control (C RNAi, solid lines) and mev-1 RNAi (dashed lines) (I) and quantification of normalized basal OCR and maximal respiration (J) (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=7-8 biological replicates from 2 independent experiments). Data information: bars represent mean ±SEM points represent mean±SEM. Across experiments, p value summary is ns=not statistical, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (K) qRT-PCR of let-721 mRNA expression in wt nematodes grown on control and RNAi expressing bacteria (n=2 biological replicates). Data information: bars represent mean ±SEM; Across experiments, p value summary is ns=not statistical, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (L-N) Normalized OCR of (L) gas-1(fc21) and (M-N) gas-1(fc21); cest-2.2 O/E nematodes fed with control and RNAi expressing bacteria (one-way ANOVA with Tukey's multiple comparison test, n=5-8 biological replicates from 2 independent experiments). Data information: bars represent mean ±SEM points represent mean±SEM. Across experiments, p value summary is ns=not statistical, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (O) Schematic representation of the proposed mechanism. In gas-1 mutants, aberrant complex I impairs the proper ETC function, resulting in diminished mitochondrial respiration. Overexpression of CEST-2.2 stimulates fatty acid beta-oxidation, which promotes TCA cycle and the transferring of electrons to ubiquinone/coenzyme Q through complex II and LET-721/ETFDH. This ultimately contributes to electron transport maintenance and ATP production.