Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

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

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 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/.

PMC Copyright notice

Extended Data Fig. 8 — a, Co-localization analysis, using confocal microscopy, of mTOR with LAMP2 (lysosomal marker) in HEK293FT cells treated with 25 μM Fasnall for the indicated times. Magnified insets and intensity plots for selected regions shown on the right. Scale bars, 5 μm. b, Quantification of co-localization from n_DMSO = 124, n_{Fasnall_30’} = 130 and n_{Fasnall_60’} = 117 individual cells. Pooled data from four independent experiments. c,d, As in a,b, but inhibiting FASN with 50 μM cerulenin for the indicated times. c, Scale bars, 5 μm. d, Quantification from n_DMSO = 103, n_{Ceru_2h} = 74 and n_{Ceru_4h} = 69 individual cells. Pooled data from three independent experiments. e,f, RagC stays lysosomal following FASN inhibition. As in c,d, but for RagC/LAMP2 co-localization. Scale bars, 10 μm. f, Quantification of co-localization from n_DMSO = 49 and n_{Ceru_4h} = 48 individual cells. Data from a representative experiment of two independent replicates are shown. g,h, Constitutively active Rags that blunt the amino-acid-starvation response do not prevent mTORC1 downregulation by FASN inhibition. g, HEK293FT cells were transiently transfected with vectors expressing FLAG-tagged RagA^QL and RagC^SN mutants, or Luciferase (Luc) as the control and treated with 25 μM Fasnall for the indicated times, or amino-acid starvation media for 1 h. h, Quantification of mTORC1 activity (p-S6K^T389/S6K), normalized to each DMSO-treated control; n = 3 independent experiments. i,j, Co-localization analysis of mTOR with LAMP2 in HEK293FT cells transiently expressing FLAG-tagged RagA^QL and RagC^SN mutants, or Luciferase (Luc) as control, and treated with 25 μM Fasnall. i, Scale bars, 5 μm. j, Quantification of co-localization from n_{Luc_DMSO} = 47, n_{Luc_Fasnall} = 50, n_{Rag_DMSO} = 52 and n_{Rag_Fasnall} = 60 cells. Data from a representative experiment of two independent replicates are shown. k, Specific staining of endogenous FASN shown by knockdown (siFASN) and immunofluorescence. Nuclei were stained with DAPI. Scale bars, 20 μm; n = 3 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.

Source data