Extended Data Fig. 4: FASN blockage or exogenous Mal-CoA downregulates mTORC1 in different mammalian cell lines.
From: Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor
a,b, Fasnall downregulates mTORC1 activity in mouse embryonic fibroblasts (MEFs.) a, Cells were treated for 30 min with 25 μM Fasnall or DMSO (−) as the control. b, Quantification of mTORC1 activity (p-S6KT389/S6K); n = 7 independent experiments. c, Cerulenin downregulates mTORC1 activity in MEFs. Cells treated for 2 or 4 h with 50 μM Cerulenin (Ceru) or DMSO (4 h) as the control (−); n = 3 independent experiments. d,e, Cerulenin downregulates mTORC1 activity in MCF-7. d, Cells were treated for 2 or 4 h with 50 μM cerulenin (Ceru) or DMSO (4 h) as the control (−). e, Quantification of mTORC1 activity (p-S6KT389/S6K); n = 9 independent experiments. f,g, FASN knockdown downregulates mTORC1 activity in MCF-7. g, Quantification of mTORC1 activity (p-S6KT389/S6K);. n = 5 independent experiments. h,i, FASN knockdown downregulates mTORC1 activity and increases Mal-CoA levels in WI-26 fibroblasts. h, Mal-K blots show total protein malonylation, indicative of intracellular Mal-CoA levels. i, Quantification of mTORC1 activity (p-S6KT389/S6K);. n = 4 independent experiments. j, Fasnall downregulates mTORC1 activity in U2OS. Cells treated for 30 min with 25 μM Fasnall or DMSO as control (−); n = 3 independent experiments. k, Cerulenin downregulates mTORC1 activity in U2OS. Cells were treated for 2 or 4 h with 50 μM cerulenin (Ceru) or DMSO (4 h) as the control (−); n = 2 independent experiments. l, Gating strategy for the flow-cytometry-based OPP incorporation assay shown in Fig. 2l. m–o, Exogenous Mal-CoA addition is capable of downregulating mTORC1. m, HEK293FT cells were treated with 25 μM Fasnall or 250 μM Mal-CoA for 30 min. n, Quantification of mTORC1 activity (p-S6KT389/S6K). o, Total protein malonylation (Mal-K blots) indicates uptake of exogenously supplemented Mal-CoA ; n = 4 independent experiments. Data in graphs are shown as the mean ± s.e.m. *P < 0.05, **P < 0.005, ***P < 0.0005. Source numerical data and unprocessed blots are provided.
