PDK1 regulates EGF-induced chemotaxis and directional migration of MCF10A cells. (A) MCF10A cells, infected with lentiviral vectors coding for a control shRNA (shScr) or for shRNAs silencing PDK1 (shPDK1 #79 and shPDK1 #81), were assayed for their migratory ability in the absence of EGF (0 ng/ml EGF), in the presence of 5 ng/ml EGF, or in the presence of 5 ng/ml EGF in both the culture well and in the insert (5 ng/ml EGF [no grad]). PDK1 silencing was verified by immunoblotting. (B) MCF10A cells were infected with a lentiviral vector overexpressing PDK1_WT compared with an empty vector and tested for PDK1 expression and cell migration. Each bar represents the mean and standard error of the mean of three independent experiments. (C) MCF10A shScr, shPDK1 #79, and shPDK1 #81 were cultured as a confluent monolayer and wounded or cultured as sparse cells. Cell movement was tracked and plotted by setting the starting value as 0 µm for both the x and y axes. (D and E) The FMI (D) and persistence (E) of >60 cells was calculated as described in the Materials and methods section. (F–H) MCF10A empty vector or PDK1_WT were tracked upon confluent monolayer wounding or during sparse cell movement (F) and their FMI (G) and persistence (H) were calculated. FMI and persistence were represented as a box plot distribution of >100 cells in three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.