Loss of WDR91 causes PtdIns3P-dependent endosomal trafficking defects. (A) Coimmunostaining of cells with EGFR and EEA1. Control and KO-91 HeLa cells were fixed and stained at the indicated time points after 25 ng/ml EGF stimulation. (B) Quantification of EGFR-EEA1 colocalization at the indicated time points after EGF stimulation. (C) Coimmunostaining of cells with EGFR and LAMP1. Cells were treated as in A. (D) Quantification of EGFR-LAMP1 colocalization in cells from C. Data are plotted as in B. (E) Confocal images of dextran 405 in control and KO-91 HeLa cells costained with EEA1 and LAMP1. Cells were incubated with dextran 405 for 30 min and then fixed immediately or after incubation without dextran for a further 1.5 h. (F) Coimmunostaining of EGFR with EEA1 (left) or LAMP1 (right) in control and KO-91 HeLa cells 60 min after EGF stimulation. Cells were treated without or with 500 nM VPS34-IN1 for 3 h. Insets show magnified views of the box in the merged images. Bars, 5 µm. (G and H) Quantification of the colocalization of EGFR with EEA1 (G) and EGFR with LAMP1 (H) as shown in F. For all quantifications, Pearson’s correlation coefficient is plotted on the y-axis. ≥50 cells were scored. Error bars represent SEM. ***, P < 0.001.