FIGURE 3:

Treatment with okadaic acid restores dephosphorylation and nuclear translocation of TFEB induced by PIKfyve inhibition. (A) HeLa cells treated with 1 µM apilimod (or DMSO vehicle control) and okadaic acid at the indicated concentration were cultured for 1 h in the growth medium and then analyzed by immunoblot using the indicated antibodies. (B) Quantitation of phospho-TFEB signal intensities from immunoblots in A, following normalization to the total TFEB protein (mean ± SD; three independent experiments). (C) HeLa cells stably expressing TFEB-GFP treated with 1 µM apilimod and 400 nM okadaic acid were cultured for 1 h in the growth medium. Cells were fixed and then analyzed by immunofluorescence microscopy. Bar, 10 µm. (D) Percentage of TFEB-GFP that is localized to the nucleus in C (mean ± SD; n > 50 cells from three independent experiments). **P < 0.01.