MRCKα mediates PDK1-induced cell migration. (A–C) MCF10A cells were tested for their migratory ability toward 5 ng/ml EGF in the presence of increasing concentrations of the inhibitor of nonmuscle myosin II Blebbistatin (A), the ROCK inhibitor Y-27632 (B), or the MRCK inhibitor chelerythrine chloride (C). (D–F) FMI of cells migrating in wound healing in the presence of increasing concentration of Blebbistatin (D), Y-27632 (E), or chelerythrine chloride (F). (G–I) MCF10A cells, stably transduced with lentiviral vectors coding for PDK1_WT or PDK1_KD, compared with an empty vector and transfected with siRNAs targeting, respectively, MRCKα, MRCKβ, or nontargeting (siCtrl), were assayed for PDK1, MRCKα, and MRCKβ protein expression (G), their migratory ability toward 5 ng/ml EGF (H), or for the directional migration in wound healing (I). (J–L) MCF10A empty vector, PDK1_WT, or PDK1_KD, transfected with siRNAs targeting ROCK1 or nontargeting (siCtrl), were assayed for PDK1 and ROCK1 protein expression (J), their migratory ability toward 5 ng/ml EGF (K), or for the directional migration in a wound healing assay (L). Each bar in A, B, C, H, and K represents the mean and standard error of the mean of seven independent experiments. In D, E, F, I, and L, data are represented as box plot distributions of >100 cells and were obtained from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.