FIGURE 6.

Inhibition of Abl kinases impairs localization of MT1-MMP and cortactin to invadopodia. A, NIH3T3-SrcY527F cells were plated on FITC-gelatin in the presence or absence of STI571 (10 μm) for 3 h. B, MDA-MB-231 cells expressing constitutively active SrcY527F were transduced with lentiviruses encoding either control or Abl/Arg miRNAs, sorted by flow cytometry, and plated on FITC-gelatin-coated cover slips for 6 h. A and B, cells were fixed and stained for MT1-MMP (red). Dark areas of gelatinase activity (black) reveal MMP-mediated degradation. Nuclei were labeled with Hoechst (blue). C, NIH3T3- SrcY527F cells expressing the indicated miRNAs were biotinylated for 30 min, lysed, and incubated with neutravidin beads. Protein complexes bound to beads were analyzed by SDS-PAGE and blotting for cell surface MT1-MMP. Total cell lysates from biotinylated samples were examined for total MT1-MMP and indicated proteins. Densitometry analysis show a ratio of surface MT1-MMP: Total MT1-MMP and were normalized to controls. D, MDA-MB-231 cells were treated with vehicle or 10 μm STI571 for 24 h. Cells were biotinylated for 30 min, lysed, and incubated with neutravidin beads. Protein complexes bound to beads were washed and analyzed by SDS-PAGE for cell surface MT1-MMP and Na,K-ATPase. Results are representative of at least three independent experiments. UB, unbiotinylated control. Densitometry shows a ratio of surface MT1-MMP: surface Na,K-ATPase normalized to controls. E, NIH3T3-SrcY527F cells (left panels) and MDA-MB-231-SrcY527F cells (right panels) were plated on FITC-gelatin-coated cover slips for 6 h, and then fixed and stained for cortactin (red), and nuclei were labeled with Hoechst (blue); dark areas of gelatinase activity (black) reveal MMP-mediated degradation.