HRS is required for the endosomal recruitment of WASH independent of VPS35. (A) HeLa cells were treated with the indicated siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting VPS35 and EEA1. Single confocal slice images. Bars: (main images) 10 µm; (insets) 2 µm. MRG, merge. (B) Pearson’s R correlation value between VPS35 and EEA1 (10 images per condition, >150 cells total). (C) Relative intensity of VPS35 on EEA1-positive endosomes normalized to endosome size (>150 cells total). (D) HeLa cells were depleted for HRS over 120 h before transfection with mCherry-WASH construct 24 h before imaging; the cells were subjected to photobleaching with a 594-nm laser. Fluorescence recovery was measured using the FRAP tool in the Slidebook image analysis suite. (E) The rate constants (k = s⁻¹) for the recovery curves were extracted using Prism. The mean rate constants of three independent experiments are plotted (>10 cells per condition per experiment). **, P < 0.01. (F) HeLa cells were depleted of HRS with siRNA over 120 h before transfection with YFP-VPS35 construct 24 h before imaging. Cells were incubated with 647-dextran for 15 min before imaging to mark the endocytic network, and YFP-VPS35–positive endosomes were subjected to photobleaching with 594-nm laser light. (G) The rate constants (k = s⁻¹) for the recovery curves were extracted using GraphPad Prism. The mean rate constants of three independent experiments are plotted (>10 cells per condition per experiment). n = 3; error bars indicate SEM.