Fig. 1.

TFEB localizes to lysosomes and accumulates in the nucleus in response to perturbation of lysosomal function. (A) Live imaging (spinning disk confocal) of TFEB-GFP (green) and DQ-BSA (red, lysosomal marker) in HeLa M cells shows an enrichment of the TFEB-GFP signal on lysosomes. Insets show higher magnification views. (B) TFEB-GFP localization without (left) or with chloroquine (CQ, 50μM, 15 hours) treatment (right). (C) Percentage of cells exhibiting lysosomal localization (p<0.01, t-test, n=3 experiments, 40 cells/condition/experiment). (D) Percentage of cells showing nuclear enrichment (p<0.01, t-test, n=3 experiments, 40 cells/condition/experiment). (E) Western blotting of total, cytoplasmic and nuclear subcellular fractions obtained from Hela M cells stably expressing TFEB-GFP +/−CQ treatment (50μM, 15 hours). Lamin A/C and tubulin represent control proteins for the nuclear and cytoplasmic fraction respectively. (F) Effect of CQ on TFEB-GFP levels (p<0.01, n=3, t-test). (G) Nuclear enrichment of TFEB-GFP +/− CQ (p<0.01, n=3, t-test). (H) Western blot for TFEB-GFP from cells grown under basal conditions +/− phosphatase treatment of the lysates. Arrows indicate the relative positions of the phosphorylated TFEB (upper arrow) and the dephosphorylated TFEB (lower TFEB). (I) Wildtype TFEB-GFP localization versus the Δ30TFEB-GFP mutant. Scale bars = 10μm