Cancerous p110α mutations promote invadopodia formation. (A) MDA-MB-231 cells stably expressing wild-type (WT), E545K, or H1047R p110α were analyzed by immunoblotting. Numbers below represent relative expression levels of the p110α constructs. (B) Cell lines stably expressing p110α were serum-starved overnight and stimulated with 8 nM EGF for 10 min. The phosphorylation status of Akt (p-Akt) was determined by immunoblotting. (C) Phase-contrast images show the morphology of the p110α cell lines. Arrowheads denote membrane protrusions. (D) Cells stably expressing the p110α constructs were cultured on fluorescent gelatin matrices for 7 h and stained with phalloidin to visualize invadopodia. Arrowheads denote the gelatin degradation sites. (E–G) Gelatin degradation activity (E), the percentage of cells with invadopodia (F), and the number of invadopodia per cell (G) were determined in p110α cell lines. (H) Cells expressing E545K or H1047R p110α were examined for gelatin degradation in the presence or absence of 100 nM PIK-75. (I) Cells expressing E545K or H1047R p110α were cultured on fluorescent gelatin matrices for 4 h and stained with anti-HA antibody to visualize localization of E545K and H1047R p110α. Insets are magnified images of the boxed regions. Arrowheads denote colocalization of the HA signals with the gelatin degradation sites. (J) Cells labeled with CellTracker green were analyzed for invasion through Matrigel-coated Transwell inserts for 24 h. Data are represented as means + SEM of seven (E), six (F–H), and three (J) independent determinations. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t tests.