myr–DGK-α promotes RCP-dependent integrin recycling, invasion, and recruitment of RCP to pseudopod tips. (A) A2780 cells were plated onto the CDM, time-lapse videos were collected in the absence (–) or presence of lyso-PA (LPA), DAG, PA, and R59022 (all at 10 µM), and pseudopod length was determined as for Fig. 3. Blue boxes indicate addition of PA. Data are represented as box and whiskers plots (black lines show medians; whiskers: 5–95 percentile); n = 3 independent experiments. ***, P < 0.0001; Mann–Whitney test. (B) A2780 cells were transfected with myr–DGK-α or pMT2 (empty vector), and the recycling of α5β1 was determined as for Fig. 1. **, P = 0.004; ***, P = 0.0008; Mann–Whitney test. (C) A2780 cells were transfected with myr–DGK-α or pMT2 together with nontargeting siRNA (siRNA control [si-con]) or an siRNA targeting RCP (si-RCP) in combination with GFP, siRNA-resistant GFP-RCP^WT (GFP-RCP^WT(resc)), or siRNA-resistant GFP-RCP^200–649 (GFP-RCP^200–649(resc)). Cells were plated onto the CDM, and pseudopod length was determined as for Fig. 3. ***, P = 0.0008; Mann–Whitney test. (D) A2780 cells were transfected with myr–DGK-α or pMT2 (empty vector) in combination with GFP-RCP. Transfected cells were plated onto CDM, and videos were captured as for Fig. 4. Representative stills are displayed. The yellow arrows indicate the direction of migration and the portion of the cell within the white square, which is presented at the bottom. Bar, 20 µm. (E–G) A2780 cells were transfected with myr–DGK-α or pMT2 in combination with either nontargeting siRNA or an siRNA targeting RCP and plated into inverted invasion assays in the presence (E and G) or absence (F) of 25 µg/ml fibronectin (FN) as indicated. Invasion was determined as for Fig. 7. Data are represented as box and whiskers plots (black lines show medians; whiskers: minimum to maximum). ***, P < 0.0001; Mann–Whitney test. Values are means ± SEM.