Fig. 3.

Impaired directional migration and motility of 4.1R^−/− keratinocytes. (A) A pseudo-wound was inflicted in a >90% confluent monolayer of keratinocytes. ‘Wound areas’ were filmed every 20 minutes for 16 hours. The representative images at 12 hours are shown. Scale bar: 50 μm. (B) The wounded area was measured using ImageJ for each representative time point. The data shown are from six experiments. Representative video clips showing the in vitro wound healing process of 4.1R^+/+ or 4.1R^−/− cells are shown in supplementary material Movie 1 and Movie 2, respectively. (C) 8-μm-diameter pore Transwell cell culture inserts were placed in six-well plates, the bottom of which was coated with fibronectin. Cells were seeded on top of the insert and incubated for 12 hours. The cells migrated to the bottom of the well were fixed and stained with Crystal Violet. Absorbance was read using a multi-well plate reader at 560 nm wavelength. The averages from three experiments are shown ± s.e.m. (D) Live-cell images were obtained every 5 minutes over a period of 3 hours (60 images in total). Each track represents an individual cell; red tracks are cells migrating negatively along the y-axis, whereas black tracks indicate cells migrating positively. The representative video clips showing the motility of an individual 4.1R^+/+ or 4.1R^−/− cell are shown in supplementary material Movie 3 and Movie 4, respectively. (E) The distance migrated was measured using the cell migration and chemotaxis plug-in (Ibidi) for ImageJ. 4.1R^−/− cells (white bars) show a significant increase in overall distance migrated. n=30 (4.1R^+/+) and n=41 (4.1R^−/−).