Figure 5.

PDK1 and Akt are essential downstream effectors of p110α for invadopodia formation. (A) MDA-MB-231 cells were transfected with control or two distinct PDK1 siRNAs for 48 h and used for immunoblotting to determine the amount of PDK1. (B–D) Cells transfected with the control or PDK1 siRNA were cultured on fluorescent gelatin-coated coverslips for 7 h. Degraded areas on the gelatin matrix (B), the percentage of cells with invadopodia (C), and the number of invadopodia per cell (D) were quantified for transfected cells. (E) Cells were transfected with control or two different sets of siRNAs targeting Akt1, 2, and 3 for 48 h and used for immunoblotting analysis with the anti–pan-Akt antibody. (F–H) Degraded areas on the gelatin matrix (F), the percentage of cells with invadopodia (G), and the number of invadopodia per cell (H) were quantified for siRNA-transfected cells. (I) Cells stably expressing E545K or H1047R p110α were transfected with indicated siRNAs for 48 h and tested for invadopodia activities for 7 h. (J) MDA-MB-231 cells plated onto fluorescent gelatin-coated coverslips for 4 h were stained with the anti-Akt or anti-PDK1 antibody. Insets are magnified images of the boxed regions. Arrowheads denote the accumulation of Akt and PDK1 signals at the gelatin degradation sites. Data are represented as means + SEM of six (B, G, and H), four (C, D, and I), and three (F) independent determinations. *, P < 0.02; and **, P < 0.005 by Student’s t tests.