Figure 2.

Notch1 cortical signaling regulates epithelial cell architecture and suppresses EGFR mitogenic signaling. (A) Individual time frames from live cell movies of actin within scramble control (SCR) and NOTCH1^KO ducts labeled with SPY650-FastAct (black). Inset for individual time frames outlined in magenta in the 0:00 (hour:min) frame. Parent cell is labeled with (*) and daughter cell is labeled with (**). Scale bar, 50 µm. (B) Fluorescence micrographs of dividing SCR, NOTCH1^KO, and ICD^KO cells in ducts labeled with Hoechst (blue) and phalloidin (white). Scale bar, 10 µm. (C) 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. (D) Quantification of spindle axis angle measured from independent cells in ducts during metaphase as shown in B. Spindle axis angle is measured relative to the basal ECM interface, with 0° denoting a parallel axis. n = 13 spindles from at least three independent ducts. (E) Quantification of the percentage of EdU positive nuclei in ducts. n ≥ 9 independent ducts. (F) YZ orthogonal projections from fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO cells labeled with phalloidin (white) and Hoechst (blue). (G) Quantification of regions of cell multilayering per field of view in fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO cells. n ≥ 10 fields of view from three independent experiments. (H) Quantification of cell height from SCR, NOTCH1^KO, and ICD^KO cells plated on hydrogels. n ≥ 10 fields of view from three independent experiments. (I) Western blot of lysates from confluent SCR, NOTCH1^KO, and ICD^KO cells cultured with high EGF (20 ng/ml) and immunoblotted for pEGFR (Y845), EGFR, and Notch1. (J) Quantification of Western blot intensity difference of pEGFR and total EGFR levels in cells stimulated with high (20 ng/ml) or low (2 ng/ml) EGF. n = 3 independent experiments. (K) Top: Fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO cells immunostained for EGFR (black). Bottom: Fluorescence EGFR (magenta) micrograph overlay with phalloidin (green). Scale bar, 10 µm. (L) Quantification of relative junctional to cytoplasmic EGFR intensity. n ≥ 20 cells from three independent experiments. (M) Representative medial confocal slice micrographs of NOTCH1^KO ducts treated with DMSO or 1 µM Erlotinib labeled with phalloidin (black). Scale bar, 100 µm. (N) Quantification of duct lumen diameter. Average duct diameters from n ≥ 15 independent ducts. (O) Quantification of the percentage of EdU-positive nuclei in NOTCH1^KO cells treated with DMSO or 1 µM Erlotinib. n ≥ 15 fields of view from three independent experiments. Western blots are representative of three independent experiments. For plots D, E, G, H, J, and L, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns denotes non-significant. For plots N and O, mean ± SEM; two-tailed unpaired t test, *P < 0.05, ***P < 0.001. Source data are available for this figure: SourceData F2.