Pharmacological inhibition of PDK1 and Akt blocks invadopodia formation. (A and B) MDA-MB-231 cells were serum-starved overnight and treated with inhibitors, 10 µM OSU-03012 for PDK1 (A) or 20 µM Akt inhibitor VIII (Akt i VIII) for Akt (B) for 1 h. The cells were subsequently stimulated with 8 nM EGF for 10 min and used for immunoblotting to determine the phosphorylation status of Akt (p-Akt). (C) MDA-MB-231 cells were cultured on fluorescent gelatin-coated coverslips for 7 h in the presence of various inhibitors, including OSU-03012, Akt inhibitor VIII, and calphostin, and GF109203X for PKC. The degraded areas on the gelatin matrix were quantified. (D and E) Dose–response curves of gelatin degradation obtained in the presence of increasing concentrations of OSU-03012 (D) or Akt inhibitor VIII (E) are shown. (F) Representative images of MDA-MB-231 cells treated with 10 µM OSU-03012 and 20 µM Akt inhibitor VIII are shown. Arrowheads denote the gelatin degradation sites. (G–J) The percentage of cells with invadopodia (G and I) and the relative number of invadopodia per cell (H and J) were quantified for cells treated with 10 µM OSU-03012 (G and H) or 20 µM Akt inhibitor VIII (I and J). (K) Cells expressing E545K or H1047R p110α were examined for gelatin degradation in the presence of 10 µM OSU-03012 or 20 µM Akt inhibitor VIII. Data are represented as means ± SEM of six (C, I, and J), four (E, G, H, and K), and three (D) independent determinations. *, P < 0.02; and **, P < 0.005 by Student’s t tests.