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. 2021 Nov 3;6:375. doi: 10.1038/s41392-021-00774-2

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© The Author(s) 2021

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. 3 — The nTCA cycle is incomplete and different from the mitochondrial TCA cycle. a HepG2 cells were immunostained with antibodies against CS, ACO2, IDH3A, OGDH, SUCLG2, FH, or MDH2. The mean immunofluorescent intensity of the indicated protein in mitochondria and nucleus was analyzed by Image J software. Error bars represent mean ± SD for triplicate experiments. b Nuclei isolated from ACO2-deficient HepG2 cells were incubated with ¹³C₆-citrate and acetyl-CoA for detection of ¹³C-labeled metabolic intermediates related to the TCA cycle by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). Western blot analysis was utilized to determine the expression of indicated protein. c Nuclei isolated from MDH2-deficient HepG2 cells were incubated with ¹³C₄-malate and acetyl-CoA for detection of ¹³C-labeled metabolic intermediates related to the TCA cycle by LC-MS. The data were normalized to protein concentration. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). Western blot analysis was utilized to determine the expression of indicated protein. d Mitochondria and nuclei isolated from an equal number of HepG2 cells were incubated with oxaloacetate and acetyl-CoA for measurement of ATP production using the ATP Colorimetric/Fluorometric Assay Kit. Error bars represent mean ± SD for triplicate experiments (*p < 0.05)