Cilengitide and osteopontin drive alterations in α5β1 recycling and cell migration that are dependent on RCP. (A) A2780 cells were transfected with a nontargeting shRNA (shRNA-CON), a short hairpin targeting RCP (shRNA-RCP), GFP-RCP^wt, or dominant-negative GFP-RCP^621E. Recycling of α5β1 integrin was determined in the presence and absence of 1 μM cilengitide as for Fig. 1 A. Values are mean ± SEM; n = 3 independent experiments. (B) Cells were transfected with GFP-RCP^wt or GFP-RCP^621E and allowed to grow to confluence. Confluent monolayers were wounded with a plastic pipette tip and the cells were allowed to migrate into the wound in the absence (Basal) or presence of 1 μM cilengitide (Cil) or 0.5 μg/ml osteopontin (Osp). The cells were observed by time-lapse video microscopy, the movement of individual cells followed using cell tracking software, and this is presented as overlays of representative trajectories described by cells during the first 9 h of their migration into the wound. The starting position of each cell is denoted by the red dot. Examples of time-lapse movies that correspond to these experiments are included as Videos 1–3 as indicated . The persistence and speed of migration were extracted from the track plots. Persistence is defined as the ratio of the vectorial distance traveled to the total path length described by the cell. Values are mean ± SEM; n = 3 independent experiments. Bar, 50 μm.