Inhibition of αvβ3 promotes migration into matrigel that is dependent on RCP and engagement of α5β1 with FN. (A) Migration of A2780 cells expressing a nontargeting shRNA (shRNA-Con) or a short hairpin targeting β3 integrin (shRNA-β3) into a matrigel plug in the presence and absence of 25 μg/ml FN was determined using an inverted matrigel plug assay. 1 μM cilengitide (CIL) and 0.5 μg/ml osteopontin (OSP) were added as indicated to the matrigel and to both the upper and lower chambers of the inverted matrigel plug assay. Invading cells were stained with Calcein AM and visualized by confocal microscopy. Serial optical sections were captured at 15-μm intervals and are presented as a sequence in which the individual optical sections are placed alongside one another with increasing depth from left to right as indicated. Migration was quantitated by measuring the fluorescence intensity of cells penetrating the matrigel to depths of 45 μm and greater and expressing this as a percentage of the total fluorescence intensity of all cells within the plug. Data represents mean ± SEM from three independent experiments. (B) The involvement of α5β1 integrin in migration driven by addition of cilengitide (left and right) or induced by suppression of αvβ3 levels (middle) was determined by addition of the indicated integrin- and FN-blocking antibodies (left and middle) and also by RNAi of the α5 and β1 subunits of α5β1 (right). Migration was quantified as for A. Values are mean ± SEM from three independent experiments. (C) The migration of A2780 cells expressing nontargeting shRNA (shRNA-CON), a short hairpin targeting RCP integrin (shRNA-RCP), GFP, GFP-RCPwt, or GFP-RCP621E into FN-containing matrigel was determined and quantified as for A.
