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. 2022 Apr 4;13:1789. doi: 10.1038/s41467-022-29426-x

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Quantitation of intracellular succinate level measured by LC-MS in resting Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB (n = 3 animals examined over two independent experiments). b Seahorse quantitation of OCR of resting Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB at the indicated time points (n = 3–4 animals examined over 5 independent experiments). c OCR components from (b) plotted as proportions of maximal OCR (after FCCP treatment) (n = 3–4 animals examined over five independent experiments). d Quantitation of the contribution of proton leakage from (b) as determined by Ant/Rot treatment (n = 3–4 animals examined over five independent experiments). e Respiratory status of Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB in (b) displayed as state_apparent (n = 3–4 animals examined over five independent experiments). f–i OCR of Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB sequentially treated with saponin (Sap), adenosine diphosphate (ADP), and the indicated substrates (red) for ETC complexes CI (f), CII (g), CIII (h) and CIV (i), as indicated. Side bar graphs show basal and substrate-induced (red) OCR. Oligo: oligomycin A; Rot: rotenone; Ant: antimycin A; TMPD: N,N,N′,N′-Tetramethyl-p-phenylenediamine; Asc: ascorbate; Pyr: pyruvate; Mal: malate (n = 4 animals examined over two independent experiments). j Quantitation of relative activity of CI in Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB as measured by colorimetric assay (n = 3–4 animals examined over two independent experiments). k Quantitation of NADH autofluorescence in Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB (gated as in Supplementary Fig. 1a) as measured by FACS (n = 4 animals examined over three independent experiments). l CI activity in Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB expressed as the ratio of NAD⁺ (CI substrate) to NADH (CI product), as measured by colorimetric assay (n = 3–4 animals examined over two independent experiments). m Representative histogram (left) and quantitation (right) of immunostaining to detect the expression of succinate dehydrogenase a (Sdha, the major subunit of CII) in Gclc^fl/fl and Gclc^fl/fl Mb1-Cre⁺ FoB (gated as in Supplementary Fig. 1a) (n = 3 animals examined over three independent experiments). For all applicable figure panels, data are mean ± SD and each dot represents one single mouse, except for (b) and (f)–(i) where each dot represents the mean of 3–5 mice. Significance (P) was calculated with unpaired t-test, except for f–i (2 way ANOVA). **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.