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. 2021 Apr 1;23(4):413–423. doi: 10.1038/s41556-021-00637-6

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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. 4 — a, Metabolite levels of 2OG and succinyl-carnitine (a surrogate for succinyl-CoA) in HUVECs transduced with control (AdCtrl) or FOXO1^A3-encoding (AdFOXO1^A3) adenoviruses (n = 4 independent samples). b, Immunoblot analysis of OGDH protein abundance in control and OGDH-depleted HUVECs. Cells were generated by lentiviral transduction of FLAG-tagged Cas9 nuclease and control (gCtrl) or OGDH-targeting (gOGDH) gRNAs. c, OCRs in gCtrl- and gOGDH-transduced HUVECs under basal conditions and in response to FCCP (n = 8 independent samples). d, 2HG metabolite levels in gCtrl and gOGDH HUVECs measured by LC–MS (n = 7 (gCtrl) and 8 (gOGDH) independent samples). e, Enantioselective LC–MS measurement of R- and S-2HG levels in gCtrl and gOGDH HUVECs (n = 7 (gCtrl) and 8 (gOGDH) independent samples). f, Cell-proliferation curves comparing gCtrl and gOGDH HUVECs (n = 12 independent samples). g, EdU incorporation in control and OGDH-deficient HUVECs. The percentage of EdU-positive ECs is shown (n = 8 (gCtrl) and 12 (gOGDH) independent samples). h, EdU incorporation in control and cell-permeable 2OG-treated HUVECs. The percentage of EdU-positive ECs is shown (n = 5 independent samples). i, Immunoblot analysis of OGDH, SDHA and FH protein levels in control (gCtrl) and CRISPR–Cas9-engineered HUVECs (gOGDH, gSDHA or gFH). j, LC–MS measurement of 2HG enantiomers showing that OGDH-deficient HUVECs (gOGDH), but not SDHA- or FH-deficient cells, have increased S-2HG levels (n = 8, 7, 8 and 8 independent samples for gCtrl, gOGDH, gSDHA and gFH, respectively). k, EdU incorporation in gCtrl, gOGDH, gSDHA and gFH HUVECs. The percentage of EdU-positive ECs is shown (n = 5, 4, 4 and 4 independent samples for gCtrl, gOGDH, gSDHA and gFH, respectively). Western blot data in b and i are from the respective experiment, processed in parallel, and are representative of at least three independent experiments. For a, c, d–h, j and k, the data represent the mean ± s.e.m.; two-tailed unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant. The numerical data, unprocessed western blots and P values are provided as source data.

Source data