Depletion of Bet1 decreases the MT1-MMP level on the ventral cell surface and alters the morphology and dynamics of MT1-MMP–positive endosomes. (A) Some of the MT1-MMP–positive dots on the ventral surface of the cells overlap gelatin degradation areas. MDA-MT1-mycHis cells were seeded onto TRITC-gelatin and then incubated on ice with anti-MT1-MMP antibodies that recognize the extracellular domain before fixation and permeabilization. Then, the cells were fixed, permeabilized, stained with secondary antibodies, and visualized by confocal microscopy. (B) Depletion of Bet1 decreases MT1-MMP dots on the ventral surface. MDA-MT1-mycHis cells were treated with Bet1 siRNAs, and then MT1-MMP–positive dots on the ventral surface of the cells were analyzed as described in A. Antibodies that recognize the extracellular domain of integrin β1 were used to confirm the ventral surface localization of MT1-MMP. (C and D) Determination of the intensity (C) and number (D) of MT1-MMP–positive dots. (E) Bet1 depletion enlarges MT1-MMP–positive endosomes. MDA-MB-231 cells treated with the indicated siRNAs were immunostained for MT1-MMP and GPP130 and then analyzed by confocal microscopy. (F and G) Quantitation of enlarged MT1-MMP–positive endosomes (F) and the dispersed Golgi (G). (H) The protein level of MT1-MMP was not affected by Bet1 knockdown. Total cell lysates were prepared from siRNA-treated MDA-MB-231 cells, and then immunoblotting for MT1-MMP and calnexin (loading control) was performed. (I and J) Enlarged MT1-MMP–positive endosomes were less dynamic. Live cell imaging of MT1-MMP–positive endosomes was performed in mock or Bet1 siRNA-transfected MDA-MT1-mCh cells. Scale bar: 20 μm in A and B; 10 μm in E; 3 μm in I and J. *, P < 0.05; **, P < 0.01; vs. mock in C, D, F, and G.