DGK-α is required for cRGDfN^MeV- and mutant p53-driven random migration on 2D surfaces. (A) A2780 cells were transfected with GFP-actin in combination with either nontargeting siRNA (siRNA control [si-con]) or an siRNA targeting DGK-α (si-DGKα) and allowed to grow to confluence on glass surfaces. Confluent monolayers were wounded with a plastic pipette tip, and the cells were allowed to migrate into the wound in the absence (−) or presence of 36 nM cRGDfN^MeV. Time-lapse videos were recorded (Videos 1, 2, and 3), and representative stills are displayed. Bar, 20 µm. (B) A2780 cells were transfected with nontargeting siRNA or an siRNA targeting DGK-α, grown to confluence, and wounded as for A. The cells were observed by time-lapse video microscopy, the movement of individual cells was followed using cell-tracking software, and representative trackplots of trajectories described by cells during the first 8 h of migration into the wound are presented. (C and D) A2780 cells were transfected with nontargeting siRNA, an siRNA targeting DGK-α, or siRNA targeting DGK-α in combination with siRNA-resistant DGK-α (si-DGKα/DGKα(resc)), and their migration into scratch wounds in the absence (−) or presence of 36 nM cRGDfN^MeV or 2.5 µM cRGDfV both with or without 10 µM R59022 or vehicle control (DMSO) was recorded as for B. The forward migration index was extracted from the trackplots. (E–G) H1299 cells expressing p53^273H or a control vector were transfected as for C and D, and their migration into scratch wounds in the presence and absence of 10 µM R59022 or vehicle control (DMSO) was recorded as for B. The forward migration index was extracted from the trackplots as for C. (C, D, F, and G) Data are represented as box and whisker plots (black lines shows medians; whiskers: 5–95 percentile); n = 3 independent experiments. ***, P < 0.0001; Mann–Whitney test.