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Figure 7


Fig. 7. Loss of {alpha}3 enhances directionality and velocity of keratinocyte migration. (A) Confluent MK{alpha}3+ and MK{alpha}3– cells were deprived of growth factors, incubated for 2 hours with mitomycin C (10 µg/ml), and scratched with the tip of a pipette prior to EGF stimulation. The black bars indicate the wound edges at t=0. Scale bar: 100 µm. Wound areas were measured using ImageJ, and the ratio of the wound area after overnight migration over the wound area at t=0 was calculated and expressed in the bar graph. Values shown represent the means ± s.e.m. of three independent experiments (***P<0.0005). (B) EGF-induced phosphorylation of ERK1/2 was monitored by western blotting in lysates of MK{alpha}3+ and MK{alpha}3– cells that were deprived of growth factors prior to EGF stimulation for the indicated time points. (C) Cells were sparsely seeded on Col-I and monitored in time-lapse recordings for 16 hours. An image was captured every 5 minutes. Cell tracks were then determined using ImageJ. The migration plots indicate tracks from ten individual cells from four independent experiments. To quantify the average velocity and directionality (D/T ratio), data from four independent experiments were pooled. The graphs represent the means ± s.e.m. from ~50 cells (*P<0.05, **P<0.005, ***P<0.0005). To determine whether polarization was stable, cells were sparsely seeded on Col-I and monitored by time-lapse recordings. An image was captured every 5 minutes. A cell was considered stably polarized when maintaining a leading lamellipodium for 1 hour. The number of polarized cells was counted and expressed as a percentage of the total number of cells. The graph shows the means ± s.e.m. from 250 cells pooled from four independent experiments (*P<0.05). (D) Cells were sparsely seeded on Ln-332 or Col-I, serum-starved, and then stimulated with 5 ng/ml EGF at the indicated time points. An image was captured every 5 minutes. Cell tracks were determined using Matlab (Mathworks), and average velocity was plotted over time. The graphs depict the average velocity of ~50 cells from a representative experiment.