Fig 1.
(A) Representative OCR curves showing the characteristic responses to the uncoupler FCCP and the mitochondrial inhibitors DCCD and sodium azide. Note that FCCP and DCCD injections are carried out separately (different plates or different wells) in C. elegans. (B) Equations and measurement timepoints used to calculate each bioenergetic parameter.
Fig 2.
Normalization parameters for different larval stages of C. elegans.
A) Larval stages and timepoints for each stage used in this study. B) Representative images of C. elegans at each stage. C) Total protein content at each stage. D) Worm volume at each stage. E) Nuclear DNA copy number at each stage. F) Mitochondrial DNA copy number at each stage. G) Mitochondrial/nuclear DNA ratio at each stage. All normalization endpoints had 3–5 biological replicates per stage. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 3.
Basal oxygen consumption rate (OCR) at different larval stages after normalization.
A) Basal OCR per worm. B) Basal OCR per mg protein. C) Basal OCR per volume. D) Basal OCR per ncDNA copy number. E) Basal OCR per mtDNA copy number. F) Basal OCR per mtDNA/ncDNA ratio. n = 3–5 biological replicates across stages. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 4.
Basal mitochondrial oxygen consumption rate (OCR) at different larval stages after normalization.
A) Basal mitochondrial OCR per worm. B) Basal mitochondrial OCR as percent total basal. C) Basal mitochondrial OCR per mg protein. D) Basal mitochondrial OCR per volume. E) Basal mitochondrial OCR per ncDNA copy number. F) Basal mitochondrial OCR per mtDNA copy number. G) Basal mitochondrial OCR per mtDNA/ncDNA ratio. n = 2–3 biological replicates across stages. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 5.
ATP-linked mitochondrial oxygen consumption rate (OCR) at different larval stages after normalization.
A) ATP-linked OCR per worm. B) ATP-linked OCR as percent total basal. C) ATP-linked OCR per mg protein. D) ATP-linked OCR per volume. E) ATP-linked OCR per ncDNA copy number. F) ATP-linked OCR per mtDNA copy number. G) ATP-linked OCR per mtDNA/ncDNA ratio. Data shown using 40 μM DCCD for larval stages L1-late-L3 and 80 μM for L4 stage. n = 2–3 biological replicates across stages. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 6.
Maximal oxygen consumption rate (OCR) at different larval stages after normalization.
A) Maximal OCR per worm. B) Maximal OCR as percent total basal. C) Maximal OCR per mg protein. D) Maximal OCR per volume. E) Maximal OCR per ncDNA copy number. F) Maximal OCR per mtDNA copy number. G) Maximal OCR per mtDNA/ncDNA ratio. Data shown using 25 μM FCCP across all stages. n = 2–3 biological replicates across stages. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 7.
Spare respiratory capacity at different larval stages after normalization.
A) Spare OCR per worm. B) Spare OCR as percent total basal. C) Spare OCR per mg protein. D) Spare OCR per volume. E) Spare OCR per ncDNA copy number. F) Spare OCR per mtDNA copy number. G) Spare OCR per mtDNA/ncDNA ratio. Data shown using 25 μM FCCP across all stages. n = 2–4 biological replicates across stages. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 8.
Proton leak at different larval stages after normalization.
A) Proton leak OCR per worm. B) Proton leak OCR as percent total basal. C) Proton leak OCR per mg protein. D) Proton leak OCR per volume. E) Proton leak OCR per ncDNA copy number. F) Proton leak OCR per mtDNA copy number. G) Proton leak OCR per mtDNA/ncDNA ratio. n = 2–3 biological replicates across stages. Different letters represent p < 0.05 with Tukey’s posthoc following ANOVA.
Fig 9.
Non-mitochondrial oxygen consumption rate (OCR) at different larval stages after normalization.
A) Non-mitochondrial OCR per worm. B) Non-mitochondrial OCR as percent total basal. C) Non-mitochondrial OCR per mg protein. D) Non-mitochondrial OCR per volume. E) Non-mitochondrial OCR per ncDNA copy number. F) Non-mitochondrial OCR per mtDNA copy number. G) Non-mitochondrial OCR per mtDNA/ncDNA ratio. n = 2–3 biological replicates across stages. Letters represent p < 0.05 with Tukey’s posthoc following ANOVA.