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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. 2 — a–c, Cerulenin boosts Mal-CoA levels and inhibits TORC1 activity (n = 4 independent experiments). a, Immunoblots of the lysates of yeast cells expressing the fapR/fapOp-yeGFP Mal-CoA reporter system treated with 20 μM cerulenin for the indicated times. GFP expression serves as an indicator of the Mal-CoA levels. Phosphorylation at T737 of Sch9 (p-Sch9^T737) was used to assess TORC1 activity. b,c, Calculated relative Mal-CoA levels (GFP/Adh1 ratio; b) and relative TORC1 activity (p-Sch9^T737/Sch9 ratio; c). d, Immunoblots of the lysates of wild-type (WT) and Acc1^S1157A-expressing yeast cells that were cultured to the exponential phase (Ctrl), starved of nitrogen (–N), or starved and restimulated with Gln for the indicated times (n = 3 independent experiments). TORC1 activity was assessed by Sch9 phosphorylation. e, Levels of the relative TORC1 activity (p-Sch9^T737/Sch9 ratio) in d. f,g, Pharmacological inhibition of FASN downregulates mTORC1. Phosphorylation of S6K at T389 and 4E-BP1 at T37 and T46 was used to assay mTORC1 activity (n = 6 independent experiments). f, Immunoblots of the lysates of HEK293FT cells treated with 25 μM Fasnall for the indicated times. g, Normalized p-S6K^T389/S6K ratio. h,i, HEK293FT cells were treated with 50 μM cerulenin for the indicated times. The levels of p-S6K^T389 were used to assay mTORC1 activity (n = 8 independent experiments). h, Mal-K immunoblots indicating total protein malonylation as a readout for Mal-CoA levels. i, Normalized p-S6K^T389/S6K ratio. j,k, HEK293FT cells were treated with control siRNA (siCtrl) or siFASN (FASN knockdown). The levels of p-S6K^T389 and p-4E-BP1^T37/46 were used to assay mTORC1 activity (n = 6 independent experiments). j, Mal-K immunoblots showing total protein malonylation. k, Normalized p-S6K^T389/S6K ratio. l, Effects of FASN inhibition on de novo protein synthesis. O-Propargyl-puromycin (OPP) incorporation assay for HEK293T cells treated with cerulenin (50 μM, 4 h) or dimethylsulfoxide (DMSO) as the control. Data represent the median fluorescence intensity of OPP Alexa Fluor 488; n = 6 biological replicates from two independent experiments. b,c,e,g,i,k,l, Data are the mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.0005. Ceru, cerulenin. Source numerical data and unprocessed blots are provided.

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