Fig. 6. Cytoplasmic localization of TFEB is restored after removal of Torin-1 and siramesine, but not after removal of sunitinib from the growth medium.

a U2OS-TFEB-eGFP cells were treated with Torin-1 (10 nM), siramesine (10 µM), or sunitinib (10 µM) for 2 hr. Following 2 hr of incubation, cells were washed with fresh medium, and incubated with drug-free medium for an additional 2 hr. During these 4 hr of incubation TFEB-eGFP was visualized by an InCell Analyzer fluorescence microscope. b The ratio between nuclear and cytoplasmic TFEB-eGFP levels was determined by staining nuclei with Hoechst 33342 (2 µg/ml) prior to drug exposure (nuclear staining is not shown), and image analysis by InCell investigator software. *p value < 0.05, **p value < 0.01—two-tailed Student's t test. Error bars denote standard deviation