Fig. 6. PAK regulates focal adhesions and cell migration. (A) Depletion of PAK kinases increases focal adhesions. Fluorescence micrographs of PAK-depleted cells and control cells. HelaS3 cells were co-transfected with siRNAs against PAK1 and PAK2 for 48 hours and subsequently plated on laminin-coated coverslips for another 48 hours. Coverslips were fixed and stained with anti-vinculin antibody and phalloidin as in Fig. 2A. Bars, 10 µm. (B) Quantification of focal adhesions was performed as in Fig. 2D. ^*P<0.01, two-tailed t-test. (C) Depletion of PAK kinases inhibits myosin light-chain phosphorylation. Cells were transfected with siRNAs targeting luciferase control (ctrl) or PAK1 and PAK2. Ninety hours after transfection, cells were lysed directly into 2x sample buffer. Levels of PAK1, PAK2 and phosphorylated MLC were analyzed by western blotting. The phosphorylated MLC blot was stripped and re-blotted with an anti-MLC antibody. Data shown are representative of three independent experiments. (D) Depletion of PAK kinases inhibits cell migration. HelaS3 cells were transfected with the indicated siRNAs for 72 hours, plated and pretreated overnight and incubated with 5 µM of ML-7 upon wounding. Migration was quantified 7 hours after wounding, as in Fig. 4A. Shown are the averages ± s.e.m. made over eight sites. ^*P<10^–5, two-tailed t-test. Data shown are representative of two independent experiments.