Role of phosphatidylinositol 3-kinase (PI3K) and c-Src in ezrin-mediated cell motility and invasion. (a) In a wound healing experiment with WTC4 cells, the PI3K inhibitor LY294002 (10 μmol/l), the c-Src inhibitor SU6656 (10 μmol/l), or the mitogen-activated protein kinase kinase (MEK) inhibitor (PD098059; 30 μmol/l), or the solvent DMSO (dimethyl sulfoxide; 10 μl/culture) was added to cultures, and wound closure was monitored up to 24 hours, as described for Fig. 3a. Representative fields photographed after 24 hours are shown. (b) The histogram shows pooled results from clones WTC4 and WTC6 in three independent wound healing experiments with the above inhibitors. Significant reduction in motility was observed in groups treated with LY294002 (P= 0.002) and SU6656 (P = 0.03). (c) WTC4 cells were set up in transwell cultures with PI3K, c-Src, or MEK inhibitors at the concentrations indicated above, and cell invasion was assessed after 36 hours. Results are expressed as the mean cell invasion ± standard deviation of at least three independent experiments. Single asterisk (*) indicates a specific reduction in invasion compared with DMSO-treated cells (LY294002, P = 0.02; SU6656, P = 0.02). (d) For analysis of Akt and Erk1/2 activation, transfected AC2M2 cell lines (see Fig. 1a) were serum starved overnight and cultured on fibronectin substratum (10 μg/ml) for 45 min. Cells were then lysed, and equal protein amounts of each cell lysate were subjected to 10% SDS-PAGE under reduced conditions. Proteins were transferred to PVP membranes, and western blotting was carried out with antibodies against Akt pS473, pan-Akt, Erk1/2 pT185/pY187 and pan-Erk1/2. (e) For c-Src analysis, cells were plated for 2 hours on fibronectin substratum and cell lysates of Triton X-100 soluble and insoluble (cytoskeletal-associated) fractions were prepared, as described in the text. Blots were probed with antibodies against c-Src pY418 and pan c-Src.