ECM degradation by exosomal MT1-MMP is dependent on Munc13-4. (A) Exosomes (100,000-g pellet) and 400 µg cell lysate were prepared from MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Munc13-4 shRNA (Munc13-4 KD) and analyzed for indicated proteins by SDS-PAGE Western blotting. Exosomes secreted basally or from cells stimulated with 1.25 µM ionomycin for 1 h were compared. (B) MDA-MB-231 cells expressing CD63-mKATE2 and MT1-MMP-pHluorin were imaged by TIRF microscopy before (0 min) and after treatment with 1.25 µM ionomycin for 5 min. (C) Control or Munc13-4 KD cells expressing MT1-MMP-pHluorin and CD63-mKATE2 or mApple-Rab11a were fixed, permeabilized to brighten pHluorin, and imaged by confocal microscopy. (D) Pearson’s correlation coefficient for pHluorin versus mKATE2 or mApple is indicated as mean values ± SE (15 cells/group from three separate preparations are shown). (E) MDA-MB-231 cells stably expressing control shRNA (Ctrl) or Munc13-4 shRNA were grown on cover slides coated with Oregon-Green-Gelatin for 16 h and labeled with phalloidin and DAPI. Zoomed images from representative cells are shown. (F) Gelatin degradation was calculated as a percentage of clearing of gelatin from the larger field per cell, shown as mean values ± SE (nine fields of view [5–25 cells]/group from three separate preparations). *, P < 0.05. Bars, 5 μm.