Fig. 1
From: mTOR-dependent phosphorylation controls TFEB nuclear export
TFEB undergoes CRM1-dependent nuclear export. a Time-lapse analysis of HeLa cells expressing TFEB-GFP and restimulated with nutrients upon 60-min starvation. Each panel shows a 3D reconstruction of a selected frame at the indicated time points using the ImageJ software. b HeLa cells stably expressing TFEB-GFP were either left untreated (Fed), starved for amino acids for 60 min (starvation), or restimulated with amino acids for 30 min in the absence or in the presence of the CRM1 inhibitor leptomycin B (5 nM) and analyzed by confocal microscopy. c HeLa cells stably expressing TFEB-GFP were plated in 96-well plates and treated as in b, analyzed by automated high-content imaging and calculated for the ratio between nuclear and cytosolic TFEB fluorescence intensity as described in the “Methods” section. Each dot represents the average nucleo/cytoplasmic TFEB fluorescence intensity ratio analyzed in several hundred cells from three different wells. Results are mean ± SEM. n > 1500 cells per condition. ***P < 0.001, two-way ANOVA. d HEK293T cells were treated as in b and analyzed by confocal microscopy. e HEK293T shown in d were analyzed to calculate the percentage of cells showing nuclear TFEB localization. Results are mean ± SEM. n > 120 cells per condition. ***P < 0.001, unpaired t-test. f HeLa cells stably expressing TFEB-GFP and transfected with siRNA directed against CRM1 (siCRM1) or with control siRNA (siCtrl) were either starved for 60 min (starvation), or starved and restimulated with amino acids for 30 min and analyzed by confocal microscopy. g Cells shown in f were analyzed to calculate the percentage of cells showing nuclear TFEB localization. n > 30 cells per condition. Results are mean ± SEM. ***P < 0.001, two-way ANOVA. Scale bars: 10 μm
