WASH localization is controlled by PI(3,5)P₂ levels. (A) Representative images from MDA-MB-231 (top) and SCC61 (bottom) showing localization of WASH (red), cortactin (green), and actin (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control. Bars, 20 µm. (B) Representative images of MDA-MB-231 cells stably expressing mRFP-Rab7 (pseudocolored blue) immunostained by WASH (red) and cortactin (green) after 2-h treatment with 800 nM YM201636 or DMS0 diluent control. Bars, 20 µm (5 µm for magnifications). (C and D) Images were analyzed for percentage of colocalization of WASH with Rab7 (C) and Rab7 area/cell (D). 2 independent experiments, n ≥ 62 cells in each condition. ****, P < 0.0001.