Extended Data Fig. 6: ILF3 controls the lysosomal localization of GATOR1 and GATOR2 to regulate mTORC1 activity.
From: Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing
a, HEK293T cells expressing Ctrl or ILF3 shRNAs were starved of amino acids for 50 min, or starved and re-stimulated with amino acids for 10 min. Cells were then immunostained with antibodies against WDR59 (red) and LAMP2 (green), co-stained with DAPI (blue) for DNA content, and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. b, HEK293T cells stably expressing GFP-tagged WDR24, with co-expression of Ctrl or ILF3 shRNAs, were treated as in a. Cells were then immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. c, HEK293T cells expressing endogenous HA-DEPDC5, with co-expression of Ctrl or ILF3 shRNAs, were treated as in a, immunostained with antibodies against HA (red) and LAMP2 (green), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. d-f, HEK293T cells stably expressing GFP-tagged NRPL2 (d), C12orf66 (e) or ITFG2 (f), with co-expression of Ctrl or ILF3 shRNAs, were treated as in a. Cells were then immunostained with an antibody against LAMP2 (red), and observed with a laser scanning confocal microscope. Scale bar of magnified insets, 5 μm. g, Quantification of the colocalization of WDR59 (a), GFP-WDR24 (b), HA-DEPDC5 (c), GFP-NPRL2 (d), GFP-C12orf66 (e) or GFP-ITFG2 (f) with LAMP2 using Pearson’s correlation coefficient. Quantification was carried out on 40 cells examined over 3 independent experiments. Data are mean ± s.d. ****P < 0.0001, n.s., not significant (unpaired two-sided Student’s t-test). Source numerical data are available in source data.