D-3 phosphoinositides are necessary for invadopodia formation. (A) MDA-MB-231 cells transfected with the GFP, GFP-Akt-PH wild-type (WT), or GFP-Akt-PH R25C mutant construct were cultured on fluorescent gelatin-coated coverslips for 7 h and imaged by confocal microscopy. Arrowheads denote degradation sites on the gelatin matrix. (B–D) Degraded areas on the gelatin matrix (B), the percentage of cells with invadopodia (C), and the relative number of invadopodia per cell (D) were quantified for transfected cells as described in the Materials and methods. (E) MDA-MB-231 cells stably expressing GFP-Akt-PH WT were cultured on fluorescent gelatin-coated coverslips for 3 h, stained for F-actin, and observed by confocal microscopy. Insets are magnified images of the boxed regions. Arrowheads denote invadopodia where GFP-Akt-PH signals were accumulated. Data in B–D are represented as means + SEM of four independent determinations. *, P < 0.01; and **, P < 0.005 by Student’s t test.