Overexpression of Bet1 elevates gelatin degradation and the protein level of MT1-MMP on the cell surface. (A) Expression levels of MT1-MMP and Bet1 proteins. Endogenous MT1-MMP and endogenous and stably expressed Bet1 proteins in the total cell lysates of parental MDA-MB-231 cells and stable cells expressing Bet1-GFP or 3xFLAG-Bet1 were examined by immunoblotting using specific antibodies. GAPDH was analyzed as a loading control. (B) Quantitation of the ratio of cells exhibiting gelatin degradation. Parental and stable cells were subjected to gelatin degradation assaying as in Fig. 1 A. (C) Flow cytometry of the MT1-MMP level on the cell surface. MT1-MMP on the cell surface was detected with an antibody to the extracellular domain of MT1-MMP without permeabilization and then analyzed by flow cytometry. MFI, mean fluorescence intensity. (D–G) Co-overexpression of Bet1 reduces the aggregation of MT1-MMP and promotes its delivery to the cell edge in HeLa cells. Representative pictures of HeLa (D) and MDA-MB-231 (F) cells transiently transfected with MT1-MMP-mCherry and FLAG-Bet1. Quantitation of the ratio of the cells with MT1-MMP aggregates (left) and at the cell edge (right) of HeLa (E) and MDA-MB-231 (G) cells. Scale bar: 10 μm. *, P < 0.05; **, P < 0.01; vs. parental MDA in B and C; vs. MT1-MMP-mCh only in E.