The MT1-MMP– EphA2 axis modulates cell morphology. (A) The expression of indicated cadherins was assessed in the breast carcinoma cell lines (n = 3). (B) Representative confocal micrographs show EphA2 and filamentous actin (phalloidin) in the 2D cell cultures. (C, left) Representative confocal micrographs show EphA2 and filamentous actin (phalloidin) in control (scr), EphA2, and MT1-MMP knockdown MDA-MB-231 cells. (C, right) Black-and-white images visualize actin cytoskeleton (top) and intercellular spaces (bottom). (D and E) Quantitative assessment of intracellular EphA2 localization (D) and intercellular spaces within the indicated MDA-MB-231 and SUM159 knockdown cells (E; error bars indicate mean ± SEM; n = 3). P-values were determined with a Mann–Whitney U test. (F) The Src kinase inhibitor PP2 impairs intracellular EphA2 localization. MDA-MB-231 cells were incubated with 5 µM PP2 for 2 h. See Fig. S2 A for details. (G and H) Soluble lysates of the invasive cells were subjected to immunoprecipitation and immunoblotting as indicated (n = 3). The asterisk indicates IgG. Note the physical interaction of MT1-MMP and EphA2 (G) and EphA2 processing in MDA-MB-231 and BT-549 cells (H). (I) MT1-MMP is required for constitutive processing of endogenous EphA2 in MDA-MB-231 cells. Control and MT1-MMP knockdown cells were incubated with soluble ephrinA1 (1 µg/ml) for 2 h followed by immunoblotting as indicated (n = 2). Arrowheads and arrows indicate the truncated ∼60- and ∼50-kD C-terminal EphA2 fragments, respectively. GAPDH served as loading control. Bars, 50 µm.