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. 2020 Aug 6;11(8):649. doi: 10.1038/s41419-020-02825-y

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© The Author(s) 2020

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 In vitro acetylation of MTHFD2 decreases its activity. MTHFD2 proteins were purified and incubated with or without 400 μm acetyl-CoA for 15 min at 30 °C before specific enzymatic assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. b, c K88Q mutation decreases MTHFD2 activity. MTHFD2 proteins were purified by immunoprecipitation from HCT116 cells b or recombinant expressed in E. coli and purified by nickel affinity chromatography c, and the enzyme activity assays were performed. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. d The site-specific K88-acetylated MTHFD2 decreases its enzyme activity. MTHFD2 and K88Ac-MTHFD2 were recombinant expressed and purified by nickel affinity chromatography and performed activity assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. e Cisplatin represses MTHFD2 activity by acetylation. HCT116 cells were transfected with Flag-tagged MTHFD2 and treated with or without Cisplatin for 24 h. Immunoprecipitated MTHFD2 activity was detected by specific enzymatic assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. f Cisplatin cannot repress MTHFD2 activity in vitro. MTHFD2 proteins were purified and incubated with or without 40 μm cisplatin for 15 min at 30 °C before specific enzymatic assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. g Molecular modeling of acetylation of K88 in MTHFD2 (left, WT; right, K88Ac) with K88 (PDB ID 5TC4) bounded with LY345899 (an inhibitor of MTHFD2 similar to substrate). The WT MTHFD2 is shown in green chains, K88Ac is shown in blue chains and LY345899 is shown in stick format. Bottom view is enlarged to show that LY345899 binds to K88 of MTHFD2.