Cortactin levels control exosome secretion. Exosome preparations purified from equal numbers of cells were analyzed. (A) Representative nanoparticle tracking analysis (NTA) traces of exosomes derived from SCC61 control (SC) and cortactin knockdown (KD1 and KD2) cells, normalized to cell number. (B) Quantification from n = 3 independent experiments. Rescue constructs indicated after the slash (/) for empty vector (control) or WT (wild-type) cortactin. OE, cortactin overexpression in parental cells. Western blots (WBs) of cortactin levels in KD and OE cells shown in Fig. S1 and of KD/rescue cells in Fig. 5. (C) Quantitation of vesicle size by NTA. (D) Representative TEM images of exosome preparations from scrambled control (Sc) and cortactin knockdown (KD1 and KD2) cells. (E) Quantification of mean exosome number per EM image. (F) Size of vesicles ≥40 nm quantitated from EM images. Quantitations in E and F from 28–30 images per condition, n = 3 independent experiments (total number of vesicles across all images = 1,146 for control, 277 for KD1, and 129 for KD2). (G and H) Representative WBs of control and cortactin-KD whole-cell lysates (WCL) and exosomes (Exo). WCLs are loaded according to equal protein, whereas exosomes are loaded according to cell number. (I) Quantitation of WB. n = 3 independent experiments. Parametric data plotted as bar graphs, mean ± SE, and analyzed for statistical significance using a Student’s t test. Nonparametric data graphed with box and whiskers plots, where the line indicates median, the box indicates 25–75th percentile and the whiskers indicate 5–95th percentile, and analyzed by Mann–Whitney U test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.