Loss of WDR91 leads to accumulation of endosomal PtdIns3P and defective early-to-late endosome conversion. (A) Colocalization of 2×FYVE-GFP with endogenous Rab7 or LAMP1 in control and KO-91 HeLa cells. Bars, 5 µm. (B) Quantification of the colocalization of endosomal/lysosomal markers as shown in A. (C) Colocalization of endogenous EEA1 with Rab7 or LAMP1 in control and KO-91 HeLa cells. Bars, 5 µm. (D) Quantification of the colocalization of endosomal/lysosomal proteins as shown in C. (E and F) Time-lapse recording of dynamic changes of the early endosomal marker 2×FYVE-mCh and the late endosomal protein GFP-Rab7 on endosomes in control (E) and KO-91 (F) HeLa cells. Cells were imaged 24 h after transfection. 0 min refers to the time point when GFP-Rab7 was fully recruited on 2×FYVE-mCh–positive endosomes. White and yellow arrows indicate the followed endosomes before and after enrichment of GFP-Rab7, respectively. Bars, 3 µm. (G) Duration of the overlap of 2×FYVE and Rab7 on endosomes as shown in E and F. (H) Colocalization of EEA1 with Rab7 in control and KO-91 HeLa cells treated without or with 500 nM VPS34-IN1 for 3 h. Bars, 5 µm. Quantification of colocalization is shown on the right. (I) Colocalization of EEA1 with Rab7 in control and KO-91 HeLa cells treated with control siRNA (siCtrl) or Beclin1 siRNA (siBeclin1). Zoom images represent magnified views of boxed areas. Quantification of colocalization is shown on the right. For all quantifications, ≥50 cells were scored. The y-axis shows the value of Pearson’s correlation coefficient. Error bars represent SEM. *, P < 0.05; ***, P < 0.001. Images shown in E and F were derived from Videos 1 and 2, respectively.