Expression of wild-type but not Y397F-FAK restores enhanced matrix-degrading invadopodia and impaired invasion in FAK-deficient MTLn3 cells. (A) MTLn3 cells stably expressing GFP, GFP-FAK (wild type), GFP-FAK-Y397F, and GFP-FAK-K454R were transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi), cultured on fibronectin–Alexa Fluor 568 gelatin coverslips, and stained with anticortactin antibody (blue). Bar, 10 µm. (B) Quantification of active invadopodia, defined as colocalizing areas of cortactin staining and matrix degradation, is expressed as the mean number of active invadopodia per cell. (C) MTLn3 cells treated as in A were assayed for their ability to invade through Matrigel-coated membranes. Relative invasion was determined by normalizing the number of cells invaded to those transfected with control siRNA. WT, wild type. Data shown are means ± SEM of three independent experiments. *, P < 0.05 by t test compared with GFP-expressing cells transfected with control siRNA.