Figure 6.
Inactivation of ILK in keratinocytes prevents directional cell migration and Rac1-GTP formation. (A) Four days after infection with AdCre (AdCre) or an empty adenovirus (No AdCre), confluent monolayers of Ilkf/f keratinocytes were scrape-wounded and cultured in serum-free growth medium and 2.5% BSA. Photographs show representative images all along the wound edges at time = 0, and after allowing the cells to migrate into the denuded areas for 16 h. Bar, 200 μm. (B) Ilkf/f keratinocytes were infected with AdCre or with an empty adenovirus (No AdCre), followed 20 h later by transfection with a vector encoding RFP-wGBD. The cells were cultured for an additional 24-h interval and photographed as they generated extensions, which resulted in directional migration in the control population (No AdCre), but not in the AdCre-infected, ILK-deficient cells. Bar, 50 μm. (C) Ilkf/f keratinocytes were infected with AdCre or an empty adenovirus (No AdCre) and cultured for 4 d in normal growth medium. The confluent monolayers were cultured in serum-free medium containing 0.25% BSA for 16 h, at which time multiple scrape wounds were produced, and cell lysates were obtained at the indicated intervals after wounding. Active, GTP-bound Cdc42 (Cdc42-GTP) and Rac1 (Rac1-GTP) were isolated in GST-PAK pull down assays. The levels of indicated total cellular proteins were also assessed by immunoblot from samples of lysates containing each 20 μg protein. The graph represents the average + SD from densitometric analysis of Rac1GTP levels normalized to abundance of total Rac1 corresponding to the same time point (n = 4, * p < 0.005).