BafA1 precludes EEA1 membrane recruitment induced by PIKfyve inhibition with apilimod.

HEK293 cells were treated with vehicle (0.1% DMSO, panels a and b) or BafA1 (15 nM) for 40 min followed by 100 nM apilimod (or 0.1% DMSO in the control) for an additional 60 min. Cells were then fixed, permeabilized, immunostained for EEA1 and observed by confocal microscope (40x objective). (A): Shown are typical immunofluorescence images for EEA1 (panels a—c) illustrating that fluorescence signals are markedly increased in cells with apilimod treatment (panels b vs. a) and dramatically diminished upon BafA1 pretreatment (panels b vs. c). (B): quantitation of the EEA1-associated immunofluorescence by ImageJ software based on randomly selected cells (30 cells/condition) from different fields in 2 separate experiments with similar results. Data are expressed as corrected integrated density of cell fluorescence (mean ± SEM) and analyzed by one-way Anova, *P<0.05; *** P<0.001. Bar, 10 μm.