Figure 2. Notch1 cortical signaling regulates epithelial cell architecture and suppresses EGFR mitogenic signaling.

(A) Individual time frames from a live cell movie of actin within a NOTCH1^KO duct labeled with SPY650-FastAct (black). Inset for individual time frames outlined in magenta in the 0:00-hour frame. Parent cell is labeled with (*) and daughter cell is labeled with (**). Scale bar, 50 μm. (B) Maximum projection micrographs of 30 μm medial stacks of SCR, NOTCH1^KO, and ICD^KO epithelial ducts labeled with phalloidin (magenta) and EdU (green). Scale bar, 30 μm. (C) YZ orthogonal projections from fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO monolayers labeled with phalloidin (grey) and Hoechst (blue). (D) Quantification of the percentage of EdU positive nuclei in ducts. n ≥ 9 ducts from three independent experiments. (E) Quantification of regions of cell multilayering per field of view in fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO monolayers. n ≥ 10 fields of view from three independent experiments. (F) Quantification of cell height from SCR, NOTCH1^KO, and ICD^KO monolayers plated on hydrogels. n ≥ 10 fields of view from three independent experiments. (G) Western blot of lysates from confluent SCR, NOTCH1^KO, and ICD^KO monolayers immunoblotted for pEGFR (Y845), EGFR, and Notch1. (H) Quantification of western blot intensity difference of pEGFR and total EGFR levels in monolayers stimulated with EGF. n = 3 independent experiments. (I) Fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO monolayers immunostained for EGFR (black). Scale bar, 10 μm. (J) Quantification of relative cytoplasmic to junctional EGFR intensity. n ≥ 30 cells from three independent experiments. (K) Representative medial confocal slice micrographs of NOTCH1^KO ducts treated with DMSO or 1 μM Erlotinib labeled with phalloidin (black). Scale bar, 100 μm. (L) Quantification of duct lumen diameter. n ≥ 15 average diameters from three independent experiments. (M) Quantification of the percentage of EdU positive nuclei in NOTCH1^KO monolayers treated with DMSO or 1 μM Erlotinib. n ≥ 15 fields of view from three independent experiments. For plots D–F, H, and J, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test, **p<0.01, ***p<0.001, ****p<0.0001. For plots L and M, mean ± SEM; two-tailed unpaired t test, ***p<0.001.