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. 2023 Aug 10;25(9):1303–1318. doi: 10.1038/s41556-023-01198-6

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© The Author(s) 2023

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,b, Exogenous expression of a hyperactive ACC1 mutant (ACC1^S79A) cooperates with FASN inhibition to downregulate mTORC1 in HEK293FT cells without influencing the response to amino-acid (AA) starvation. The cells were transfected with vectors expressing FLAG-tagged WT ACC1, ACC1^S79A or Luciferase (Luc; as the control) and treated with Fasnall (25 μM, 30 min) or AA-starvation medium (1 h) as indicated (n = 3 independent experiments). a, Phosphorylation of S6K at T389 was used to assay mTORC1 activity. b, Levels of mTORC1 activity (p-S6K^T389/S6K ratio). c–e, ACC1 knockdown partially restores the increase in Mal-CoA levels and rescues the downregulation of mTORC1 caused by silencing of FASN. siCtrl, control siRNA; and siACC1, siRNA to ACC1. c, Immunoblots of HEK293T cell lysates. d, Calculated levels of mTORC1 activity (p-S6K^T389/S6K ratio; n = 5 independent experiments). e, Levels of Mal-K (Mal-K/tubulin ratio; n = 3 independent experiments). f, Lack of detectable mTOR malonylation in HEK293FT cells. Endogenous mTOR (left) and FASN (right) proteins were immunoprecipitated from control (−) or cerulenin-treated (50 μM, 4 h) cells. g, Tor1 is not malonylated in yeast cells. Amino (N)-terminally HA-tagged Tor1 or C-terminally HA-tagged Fas1 was immunoprecipitated from control (−) or cerulenin-treated (10 μM, 2 h) cells cultured to the exponential phase; α-HA, anti-HA. f,g, Protein malonylation was assessed using anti-Mal-K (n = 3 independent experiments); IP, immunoprecipitate; and exp., exposure. b,d,e, Data are the mean ± s.e.m. *P < 0.05; **P < 0.005; ***P < 0.0005; and NS, not significant. Ceru, cerulenin. Source numerical data and unprocessed blots are provided.

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