Extended Data Fig. 3. FASN inhibition/knockdown decreases mTORC1 activity towards multiple substrates, without affecting FASN/ACC1/mTOR levels or mTORC1 stability.
a–c, Fasnall downregulates mTORC1 activity towards Grb10 and TFEB. Cells were treated for 30 min with 25 μM Fasnall or DMSO (−) as the control. b, Quantification of Grb10 phosphorylation (p-Grb10T389/Grb10). c, Quantification of TFEB phosphorylation (p-TFEBS211/TFEB); n = 3 independent experiments. d–g, Cerulenin downregulates mTORC1 activity towards Grb10, TFEB and 4E-BP1. d, Cells were treated for 4 h with 50 μM cerulenin or DMSO (−) as the control. e, Quantification of Grb10 phosphorylation (p-Grb10T389/Grb10); n = 3 independent experiments. f, Quantification of TFEB phosphorylation (p-TFEBS211/TFEB); n = 4 independent experiments. g, Quantification of 4E-BP1 phosphorylation (p-4E-BP1T37/46/4E-BP1); n = 3 independent experiments. h–j, As in a–c, but with transient FASN knockdown. nGrb10 = 4 and nTFEB = 3 independent experiments. k–n, FASN, ACC1 and mTOR total protein levels are not affected by short-term FASN inhibition (4 h cerulenin). k, Immunoblots for the indicated proteins. l–n, Quantification of relative FASN (l), ACC1 (m) and mTOR levels (n); n = 5 independent experiments. o, Stability and composition of mTORC1 is not affected by FASN inhibition with cerulenin (4 h); n = 2 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.