Cortactin affects MVE trafficking downstream of biogenesis. (A) Representative TEM images of 50-nm cell sections for SCC61-control (Sc, left) and cortactin-KD (KD, right) cells. MVEs (seen in zooms) from control (1) and cortactin-KD cells (2 and 3). V, empty vacuole like structure. Example LE/Lys hybrid organelle shown in zoom 4. Bars, 500 nm. (B) Representative confocal micrographs of SCC61 control (Sc) and cortactin-KD1 (KD1) cells transiently transfected with GFP-Rab5Q79L (green) and stained for endogenous CD63 (purple) along with actin filaments (rhodamine phalloidin, red). Insets show 4× zooms. Bar, 15 µm. (C) Quantitation of percent GFP-Rab5Q79L positive endosomes filled with CD63. n ≥ 17 cells per condition n ≥ 3 independent experiments. Lines show mean values. (D) Representative confocal images of immunostained CD63. Bar, 10 µm. Insets (4× zooms). a and b are two example endosomes in a KD cell. (E) Quantitation of CD63-positive area/cell area. (F) Quantitation of the number of large (>1 µm) CD63-positive endosomes per cell. n ≥ 31 cells per condition n ≥ 3 independent experiments. Bar graphs represent mean ± SE. ns, not significant; *, P < 0.05; **, P < 0.01. Student’s t test used to determine statistical significance.