Extended Data Fig. 1: Genome-wide CRISPR/Cas9 knockout screening to identify potential regulators of mTORC1-dependent amino acid sensing.
From: Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing
a, HEK293T cells were starved of amino acids for different times as indicated, or starved for 2 hours and re-stimulated with amino acids for different times. Cell lysates were immunoblotted to detect the (phosphorylated) level of the indicated proteins. b, HEK293T cells were cultured with fresh complete medium, or medium lacking amino acids for 2 hours followed with or without amino acid re-stimulation for 1 hour. Cells were then immunostained with an antibody against pS6 (green), co-stained with DAPI (blue) for DNA content, and observed with a laser scanning confocal microscope. Scale bar, 100 μm. c, HEK293T cells were cultured with fresh complete medium, or medium lacking amino acids for 2 hours with or without amino acid re-stimulation for 1 hour. Cells were then immunostained with an antibody against pS6 (Alexa Fluor® 488 conjugate) and subjected to FACS-based analysis. d, The list of known regulators of mTORC1-dependent amino acid sensing identified by our CRISPR screen. e, Gene ontology (GO) analysis of the candidate genes. The top 15 enriched biological processes are shown. f, Protein-protein interaction analysis of the candidate genes. Only clustered genes are shown. Blue and red circles represent genes from the screen groups -AAs and +AAs, respectively. Unprocessed blots are available in source data.
