(a–c) qPCR analysis of Prkc iota (a), Prkc zeta (b) or NGN3 (c) in lysates from cultures of hESC-derived NGN3+ cells (EPd4—protocol A), after 24 h siRNA knockdown (KD) of aPKC iota (Prkc i KD), aPKC zeta (Prkc z KD) or aPKC iota + aPKC zeta simultaneously (Prkc i+z KD), compared to Ctrl. n = 6, 6, 6, 3 wells of cells (from left to right), from two independent experiments, (a) Ctrl/Prkci p TT < 0.0001, Ctrl/Prkcz p TT = 0.4520 (n.s.), Ctrl/Prkci + z p TT = 0.0055. (b) Ctrl/Prkci p TT = 0.7266 (n.s.), Ctrl/Prkcz p TT < 0.0001, Ctrl/Prkci + z p TT < 0.0001. (c) Ctrl/Prkci p TT = 0.0377, Ctrl/Prkcz p TT = 0.0453, Ctrl/Prkci + z p TT = 0.00177. (d) Quantification of the relative number of NGN3 + cells in cultures of EPd4 cells (protocol A), after 24 h siRNA knockdown (KD) of aPKC iota (Prkc i KD), aPKC zeta (Prkc z KD) or aPKC iota + aPKC zeta simultaneously (Prkc i+z KD) were compared to Ctrl. n = 3 wells of cells, from two independent differentiations, Ctrl/Prkci p TT = 0.6861, Ctrl/Prkcz p TT = 0.5007, Ctrl/Prkci + z p TT = 0.4540. (e) Representative cultures of hESC-derived NGN3 + cells (EPd3—Protocol A), stained for E-cadherin (E-cad;red), GFP (green) and DAPI (blue). Inset shows magnification of indicated region. (f) Representative cultures of hESC-derived NGN3 + cells (EPd3—Protocol A), stained for F-actin (red), GFP (green) and DAPI (blue). Inset shows magnification of indicated region. (g) Representative 3D-reconstruction of z-stack of a culture of hESC-derived NGN3 + cells (EPd3—Protocol A), stained for ZO-1 (red), GFP (green), Ezrin (white) and DAPI (blue). Inset shows magnification of indicated region. Right panel represents the culture viewed from the side. (h–j) qPCR analysis of aPKC iota (h), Muc1 (i) and Hes1 (j) in flow cytometry isolated Ctrl, LY-, aPKCi- or EGFRi-treated (24 h) hESC-derived NGN3 + cells (EPd4—protocol A). n = 3 extracts of RNA (from 1 differentiated plate each), from three independent differentiations, (h) Ctrl/LY p TT = 0.0444, Ctrl/aPKCi p TT = 0.0238, Ctrl/EGFRi p TT = 0.0437 (i) Ctrl/LY p TT = 0.0196, Ctrl/aPKCi p TT = 0.0013, Ctrl/EGFRi p TT = 0.0257 (j) Ctrl/LY p TT = 0.0178, Ctrl/aPKCi p TT = 0.0392, Ctrl/EGFRi p TT = 0.424. (k–m) Whole lysates from EPd10 cultures (protocol A), treated with LY, aPKCi or EGFRi for 7 days, were analyzed for the expression of Ezrin and aPKC using western blot. alpha-tubulin was used as loading control. Relative Band intensity show that LY and EGFRi treatment increase the expression of Ezrin and aPKC, whereas aPKCi treatment decreases them. n = 2. Error bars represent ± SEM. Scale bars, 50 μm (e–g). Statistic source data are found in Supplementary Table 3. Statistical analysis: two-tailed student’s t-test (p TT ).