PI4KIIs expression is required for endosomal cargo recycling. (A) FM of siCTRL- (left panels) or siPI4KIIs- (right panels) treated Hela cells pulsed with Tf-A488 (green) and chased at indicated times (0, 20, 40 min), before fixation and labeling of nuclei (DAPI, blue). Arrowheads point to perinuclear accumulation of Tf⁺ endosomes in siPI4KIIs-treated cells during chase. (B) Quantification of the average fluorescence intensity of Tf/cell at 0 min of chase (siCTRL, n = 50 cells; siPI4KIIs = 46 cells). (C) Quantification of the average intensity of Tf/cell at different time points of chase (siCTRL cells: 0 min, n = 50; 20 min, n = 58; 40 min, n = 49; siPI4KIIs cells: 0 min, n = 46; 20 min, n = 56; 40 min, n = 46) normalized relative to the 0 time point. Cell peripheries are delimited by yellow lines. Data from three independent experiments are presented as the mean ± SEM. (B and C)Two-tailed unpaired t test; ns, not significant. ***, P < 0.001; ****, P < 0.0001. Scale bars: 10 μm.