EphA2 and MT1-MMP are coexpressed in invasive breast carcinoma cells and regulate collagen invasion. (A) EphA2 is a positive regulator of MT1-MMP activity. Charts show relative pro-MMP2 activation in cells overexpressing 11 Eph receptors as quantified by gelatin zymography in our primary genome-wide gain-of-function kinome screen for MT1-MMP activity (for each kinase, n = 1; Sugiyama et al., 2010a). The relative value of mock control has been set to zero. PMA treatment served as a positive control. (B) Relative expression of MT1-MMP, EphA2, and ephrinA1 mRNAs was assessed by qPCR in the indicated human breast carcinoma cell lines (error bars indicate mean ± SEM, n = 3). (C) The corresponding protein expression and EphA2 tyrosine phosphorylation were assessed as indicated (n = 3). The arrowhead indicates truncated EphA2. GAPDH served as a loading control. (D) Light micrographs of collagen cross sections visualize the H&E-stained noninvasive breast carcinoma cells. Broken lines indicate the surface of collagen gels. Bar, 20 µm. (E) Quantification of collagen invasion of the breast carcinoma cell lines (error bars indicate mean ± SEM, n = 3). (F) Light micrographs of collagen cross sections visualize the H&E-stained invasive cells after EphA2 knockdown (EphA2-1 shRNA). Bar, 20 µm. (G) Relative invasion of EphA2 and MT1-MMP knockdown cells (error bars indicate mean ± SEM, n = 3). Invasion of the control cells (scr shRNA) was set to one. (H) Immunoblotting for MT1-MMP and EphA2 in the stable knockdown cells (n = 3). Mean values of MT1-MMP normalized with tubulin (loading control) are indicated below each blot. (I) EphA2 silencing reduces Src activation in BT-549 and MDA-MB-231 cells. Immunoblotting for the phosphorylated (pSrc; Tyr416) and total Src in the invasive cells (n = 3) is shown.