Fig. 3

The kinetics of TFEB nuclear export are modulated by nutrients via a nuclear export signal (NES). a Cross-species and b intra-family sequence alignment of a CMR1 consensus sequence located in the N-terminus of TFEB. Φ: hydrophobic residue. c HeLa cells were transfected with either WT or NES-mutant TFEB, subjected to starvation/refeeding and analyzed by confocal microscopy. d Cells described in c were analyzed to calculate the percentage of cells showing nuclear TFEB localization. n > 20 cells per condition. Results are mean ± SEM. ***P < 0.001, two-way ANOVA. e Representative time-lapse images of HeLa cells transfected with either wild-type TFEB-GFP or with TFEB mutants M144A, L147A, and I149A. Twenty-four hours after transfection, cells were imaged for the indicated time points upon photobleaching of cytosolic TFEB in FRAP experiments. f Cells described in e were analyzed and plotted for the decay in TFEB nuclear fluorescence. Results are mean ± SEM. n > 12 cells per condition. g Schematic experimental scheme and representative time-lapse images of HeLa cells stably expressing TFEB-GFP, treated as indicated and imaged for the indicated time points in FLIP experiments. h Cells described in g were analyzed and plotted for the decay in TFEB nuclear fluorescence. Results are mean ± SEM. Scale bars: 10 μm