Expression of Akt constructs affects invadopodia formation. (A) MDA-MB-231 cells stably expressing HA-tagged wild-type (WT), kinase-dead (KD), or myristoylated constitutively active (Myr) Akt1 were analyzed by immunoblotting. (B–D) Cells stably expressing the Akt constructs were cultured on fluorescent gelatin-coated coverslips for 7 h and stained for F-actin. Degraded areas on the gelatin matrix (B), the percentage of cells with invadopodia (C), and the number of invadopodia per cell (D) were quantified. Data are represented as means + SEM of six (B) and four to eight (C and D) independent determinations. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 by Student’s t tests. (E) Representative images of cells expressing the Akt constructs. Arrowheads denote the gelatin degradation sites. (F) Cells expressing WT or Myr Akt1 were cultured on fluorescent gelatin matrices for 3 h and stained with anti-HA antibody and phalloidin. Insets are magnified images of the boxed regions. Arrowheads denote localization of the HA signals at invadopodia.