Inhibition of PI(3,5)P2 production leads to accumulation of cortactin and actin at LE membrane. (A) Representative images showing cortactin (green) and actin (red) localization on Rab7+ endosomes (blue) after 2-h treatment with 800 nM YM201636 or DMSO diluent control in control and cortactin–KD MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (B) Immunoblot of cortactin expression and β-actin loading control in MDA-MB-231 cells. (C–E) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7 or Rab7 area per cell. 3 independent experiments, n ≥ 55 cells in each condition. (F and G) Number and intensity of actin dots on Rab7+ endosomes. More than 170 vesicles from 12–19 cells analyzed for each condition. Data shown as box and whiskers plots, with the box indicating the 25th and 75th percentiles, solid line indicating the median, and whiskers indicating the 95% confidence intervals. ****, P < 0.0001.
