PDK1 regulates 3D invasion through MRCKα in a kinase-independent manner. (A–C) Representative spheroids of MCF10DCIS.com cells infected with empty vector (A) or vector overexpressing PDK1_WT (B) or PDK1_KD (C), and stained with Phalloidin (red) and DAPI (blue). Spheroids are shown as confocal equatorial sections. Bars, 50 µm. (D) Maximum projection of deconvolved confocal multistack of a PDK1_WT overexpressing spheroid and enlargement (taken from the boxed region). Bars: (left) 50 µm; (right) 10 µm. (E) Immunoblot showing expression levels of MRCKα and PDK1 in MCF10DCIS.com cells infected with empty vector and vectors overexpressing PDK1_WT or PDK1_KD and stably silenced with lentivirus coding for shRNA targeting MRCKα (shMRCKα) and compared with the shScr vector. (F) Percentage of invading spheroids per number of total spheroids. (G) Mean number of invasive cells per spheroid. (H) Immunoblot showing PDK1 and ROCK1 expression levels in MCF10DCIS.com infected with the empty vector or vectors overexpressing PDK1_WT or PDK1_KD, and stably silenced with lentivirus coding for shRNA targeting ROCK1 (shROCK1) and compared with the shScr vector. (I) Percentage of invading spheroids per number of total spheroids. (J) Mean number of invasive cells per spheroid. At least 200 spheroids were evaluated for each condition. Statistical significance was calculated based on four independent experiments with Fisher’s exact test (F and I) and a Wilcoxon-Mann-Whitney test (G and J). *, P < 0.05; **, P < 0.01; ***, P < 0.001. (K) Graphical representation of the proposed model. EGF stimulation causes PI3K activation and PIP3 production at the plasma membrane. PDK1, which is localized in the cytoplasm in quiescent cells, rapidly translocates to the plasma membrane upon EGF stimulation and activates MRCKα in lamellipodia. This results in increased MyPT1 and MLC2 phosphorylation, determining myosin activity and lamellipodia retraction.