Supplementary Figure 4: Inhibition of PI3K or EGFR-signaling increase lumen formation and decreases β-cell differentiation.

(a) Representative 3D-projections of E11.5 + 7 Ctrl, LY- or EGFRi-treated explants, stained for E-cad (Outlined;Not shown), Muc1 and Insulin (Ins). (b) Luminal volume in E11.5 + 7 Ctrl and LY-treated explants. n = 4 explants (two litters), p = 0.0084. (c,d) Ngn3/Ecad mm² quantification (c), and mean apical volume quantification (Ngn3+ cells) (d) in E11.5 + 3 Ctrl and LY-treated explants. (c) n = 3 explants from each condition (two litters), p = 0.0101; (d) n = 3 explants (Total number of cells: 34 Ctrl, 39 LY), p_TT = 0.0076. (e) β-cell differentiation (Volume Insulin) quantification in E11.5 + 7 Ctrl and LY-treated explants. n = 3 Ctrl, 4 LY explants (two litters), p_TT = 0.0004. (f,g) Representative flow cytometry plots and quantification of %Ngn3^High cells in E11.5 + 3 Ngn3^{Y FP+} Ctrl, LY- or EGFRi-treated explants. n = 6 Ctrl, 5 LY, 3 EGFRi explants, from two independent experiments, Ctrl/LY p_TT = 0.0018, Ctrl/EGFRi p_TT = 0.0131. (h) Representative sections of E11.5 + 7 Ctrl, LY- or EGFRi-treated explants, stained for E-cad, Glucagon (Gluc) and DAPI. (i,j) α-cell (Glucagon +) differentiation quantifications in E11.5 + 7 Ctrl, LY- (i) or EGFRi-treated (j) explants. LY: n = 3 explants, from two litters, p_TT = 0.7691; EGFRi: n = 3 explants (two litters), p_TT = 0.6728. (k) Luminal Volume quantification in E11.5 + 7 Ctrl and EGFRi-treated explants. n = 7 Ctrl, 5 EGFRi explants, from three litters, p_TT = 0.0351. (l) Ngn3/Ecad mm² quantification in E11.5 + 3 Ctrl and EGFRi-treated explants. n = 5 explants, from three litters, p_TT = 0.0224. (m) β-cell differentiation (Volume Insulin) quantification in E11.5 + 7 Ctrl and EGFRi-treated explants. n = 5 Ctrl, 8 EGFRi explants, from three litters. p_TT = 0.0073. (n,o) Relative band intensity quantifications of Rac1 (n) and p85 (o) on western blots in Fig. 3i. Rac1: n = 5 protein samples, from two litters, p_TT = 0.0125; p85: n = 4 Ctrl, 3 EGFRi protein samples, from two litters, p_TT = 0.0438. (p) Representative flow cytometry plots and quantification of %Ngn3^High cells in E12.5 + 3 Ngn3^{Y FP+} Ctrl and aPKCi-treated explants. n = 4 explants (two litters), p_TT = 0.0059. (r) Representative 3D-projections of E12.5 + 7 Ctrl, aPKCi- or CpE-treated (from E12.5 + 4 and onwards) explants stained for E-cad (Outlined;Not shown), Muc1 and Insulin (Ins). Error bars represent ± SEM. Scale Bars, 200 μm (a), 40 μm (h), 100 μm (p). Statistic source data are found in Supplementary Table 3. Statistical analysis: two-tailed student’s t-test (p_TT).