KD of PIKfyve expression leads to accumulation of cortactin and actin at LE membrane. (A) Representative images of endogenous cortactin (red) localized at discrete subdomains of hVac14-EGFP⁺ (green) vesicular structures in SCC61 (left) and HeLa (right) cells. Bars: (main panels) 20 µm; (enlarged views) 3 µm. n = 3 independent experiments for each cell line. (B) Representative images of mCherry-ML1N*2 (red) and endogenous cortactin (blue) localization to EGFP-Rab7⁺ (green) endosomes. Small white boxes are enlarged on the right. Bars, 20 µm (3 µm for magnifications). (C) Magnifications of boxed areas labeled with asterisks in B show colocalization of mCherry-ML1N*2 (red) with EGFP-Rab7 (green). Bars, 5 µm. (D) Immunoblot of PIKfyve expression in PIKfyve-KD MDA-MB-231 cells. NTC, nontargeting control. (E) Immunofluorescence of PIKfyve siRNA-treated cells using antibodies recognizing cortactin (green), actin (red), and Rab7 (blue). Bars, 20 µm (3 µm for magnifications). (F) Percentage of colocalization of cortactin or actin with Rab7. 3 independent experiments, n ≥ 61 cells in each condition. ****, P < 0.0001.