Effects of PDGF-AA and EIPA in wound-healing assays on growth-arrested NIH3T3 fibroblasts. (A) Trajectories (normalized to common starting points) of growth-arrested NIH3T3 cells with and without 50 ng/ml PDGF-AA or 10 µM EIPA, as indicated. The radii of the red circles illustrate the mean translocation of the cells within a 5-h time period (n = 17–25). (B) Translocation (in micrometers and calculated as the distance between the position of the cell center at the beginning and at the end of the experiment) in the absence or presence of PDGF-AA or EIPA as indicated. (A and B) Data are individual trajectories (A) or means ± SEM (B) of 23–26 cells in three independent experiments. Data were analyzed using parametric or nonparametric ANOVA, and the level of significance is shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) Images of the left side of the wound in a confluent monolayer of growth-arrested NIH3T3 cells (serum starved for 24 h) at time 0 and 4 h after the wound was made. The arrows indicate the x and y directions of movement, which were used for estimating directional migration. (D) Velocity of migration perpendicular to the wound (x direction) in PDGF-AA–stimulated, growth-arrested NIH3T3 cells in the absence (open circles) or presence (closed circles) of EIPA. Directionality of movement was estimated by plotting the mean of the distance covered by each cell in the x direction as a function of time. The slope of this line is the velocity in x, in micrometers/minute, which is taken as a measure of directionality. In PDGF-AA–stimulated, growth-arrested cells, the velocity of movement into the wound is 0.104 ± 0.004 µm/min, and in the presence of 10 µM EIPA, this value is reduced to 0.028 ± 0.001 µm/min. Data shown are means ± SEM of 23–26 cells in three independent experiments. The velocity in the presence of EIPA is significantly different from that in the absence of EIPA (P < 0.0001). Ctrl., control. (E) NIH3T3 cells were serum starved for 24 h to induce growth arrest, exposed to 5 µM EIPA for 0, 1, or 6 h as indicated, and treated with 50 ng/ml PDGF-AA for 5 min as indicated (+). Cells were lysed, and Western blots of total and Tyr⁷⁵⁴-phosphorylated PDGFR-α were performed as described in Materials and methods. β-Actin was used as a loading control. Data shown are representative of three independent experiments.