FAK negatively regulates invadopodia formation and dynamics. (A) Cell lysates from MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi A and FAKsi B) were analyzed by Western blotting and probed for FAK or Pyk2. Actin was probed as a loading control. (B) MTLn3 cells were plated on fibronectin-coated coverslips and stained with anticortactin antibody (green) and rhodamine-phalloidin (red). Boxed regions depict regions of invadopodia shown magnified in insets. (C) Quantification of cortactin- and actin-containing invadopodia is expressed as the mean number of invadopodia per cell. (D) GFP-cortactin was transiently cotransfected with control siRNA or FAK siRNA (FAK siRNA B is shown) into MTLn3 cells. Cells were plated on fibronectin-coated glass-bottomed dishes and analyzed by time-lapse fluorescence microscopy. Time-lapse montages demonstrate representative images of the dynamics of the invadopodia marker GFP-cortactin over a period of 10 min. (E) Rate constants for assembly and disassembly were calculated from plots of fluorescence intensities of GFP-cortactin as described in Materials and methods (Videos 1–3). (F) Representative x-z confocal images of invadopodia in MTLn3 cells show cortactin-containing invadopodia (red) projecting into the gelatin matrix (green). Arrows indicate representative invadopodia. Data shown are means ± SEM of three independent experiments. *, P < 0.05 by t test compared with control siRNA. Bars, 10 µm.