Figure 2(A) Representative oxygen consumption rate (OCR) curve of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes as measured with a Seahorse XF24 Analyzer. Injection time of FCCP and NaN3 is indicated with arrows (n=9 biological replicates, from 3 independent experiments). Data information: points represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (B-C) Indices of (B) normalized basal OCR and (C) maximal respiration of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes across (n=9, one-way ANOVA with Tukey's multiple comparison test, n=9 biological replicates, from 3 independent experiments). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (D) Relative ATP content of gas-1(fc21) and gas-1(fc21); cest-2.2 O/E nematodes (unpaired t-test, n=3 biological replicates). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (E-F) Representative (E) Seahorse experiment and statistical analysis of (F) basal OCR and maximal respiration of 5-day-old gas-1; cest-2.2 O/E mutant C. elegans fed with control and cest-2.2 RNAi since hatching (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=7-9 biological replicates, from 3 independent experiments). Data information: , points represent mean± SEM bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (G) OCR profiles of wt, cest-2.2 O/E and cest-2.2(lf) nematodes upon sequential treatment with FCCP and NaN3 (n=9-10 biological replicates, from 3 independent experiments). Data information: points represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (H) Representative images of control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E nematodes expressing mitochondria-targeted GFP in the intestine (scale bar= 5 μm). (I) Representative high-resolution confocal images of C. elegans expressing mitochondria-targeted GFP in the body wall muscle cells (scale bar= 10 μm). Quantification of mitochondrial morphology (elongation) is reported in the right panel (one-way ANOVA with Holm-Sidak's correction, n=37-47 animals from 3 independent experiments). Data information: , middle line represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (J) Representative western blot analysis and densitometry of zcIs13[hsp-6p::GFP] expression in control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E animals (one-way ANOVA with Holm-Sidak's correction, n=3 biological replicates). Data information: , bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (K-L) Representative (K) Seahorse profile and (L) statistical analysis of basal OCR and maximal respiration of mutant nematodes (unpaired t-test, n=12-13 biological replicates, from 3 independent experiments). Data information: points represent mean± SEM , bars represent mean± SEM; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (M) Immunoblot and statistical analysis of zcIs13[hsp-6p::GFP] expression in control and nuo-6(qm200) animals fed with empty vector (C RNAi) and cest-2.2 RNAi bacteria (unpaired t-test, n=3 biological replicates). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.