Supplementary Figure 4: Additional analysis on Crb and Notch signaling.

(A) Quantification of apical membrane localization of Crb in cysts at different time points. Pearson colocalization coefficients were calculated by staining Podxl and Crb3 at different time points. Data represented are averaged Pearson coefficients ± s.d. (n = 5 averaged colocalization coefficients from 10 cells per condition, selected from 2 independent experiments; ^∗P < 0.05 (Student’s t test)). (B) Quantification of linear correlation of Crb3 and ZO1 linear profiles. Linear intensity profiles were drawn at the apical plasma membrane and across 4 tight junctions. Data represented are averaged linear (monodimensional) Pearson correlation coefficients ± s.d. (n = 6 linear profiles taken from 3 cysts per condition, randomly selected from 3 independent siRNA experiments; ^∗P < 0.05 (Student’s t test)). (C) Example ZO1 and Crb3 linear profiles of control-transfected and PLLP siRNA-transfected cysts. Red line, ZO1; green line, Crb3. Data are represented as % of maximum signal intensity (for each channel). TJ, position of tight junction (determined by peak in ZO1 staining). (D) Western blot of ligand-mediated Notch-1a transactivation assays in MDCK-II/OP9 cocultures. MDCK cells stably expressing Notch1a-myc are polarized over a monolayer of mesenchymal OP9 cells stably expressing GFP or Jagged-1 or Delta-like 1. Activated Notch1a is quantified using anti-Cleaved Notch1a (Val1744). Tubulin is used as loading control. (E) MDCK coculture system to analyse Notch1 transactivation by Jagged-1. Stable MDCK cell lines expressing Notch1 and Jagged-1 are mixed (4:1) and seeded to grow polarized cysts for 48 h. (F) Western blot of ligand-mediated Notch-1a transactivation assays in MDCK cocultures. MDCK cells stably expressing Notch1a-myc are mixed (4:1) with GFP or Jagged-1 expressing MDCK cells and grown in cysts (100,000 cells/ml) for 48 h. Activated Notch1a is quantified using anti-Cleaved Notch1a (Val1744). Tubulin is a loading control. (G) Effect of PLLP-KD or EpsR-KD in Notch1 transactivation by Jagged-1. MDCK cells stably expressing Notch1 or Jagged-1 were transfected with control, PLLP or EpsR siRNA, mixed as indicated and seeded to grow polarized cysts for 48 h. Cleaved Notch1 protein levels were analysed by Western blot. GAPDH is a loading control. (H) Quantification of Notch1 transactivation by Jagged-1 on PLLP-KD or EpsR-KD. Bars are colour coded to indicate control (black), PLLP (white) or EpsR (grey) silencing in either Notch (N) or Jagged-1 (J) MDCK cells. Data are expressed as mean ± s.d. as % of control (n = 5 extracts from 5 independent experiments; ^∗P < 0.05 (Student’s t test)).