Figure 3.

Notch1 cortical signaling stabilizes adherens junctions and cortical actin. (A) Super-resolution by optical pixel reassignment (SoRa) fluorescence micrographs of scramble control (SCR), NOTCH1^KO, and ICD^KO cells immunostained for E-cadherin (black). Scale bar, 10 µm. (B) Quantification of the frequency of focal adherens junctions. n ≥ 12 fields of view from three independent experiments. (C) SoRa fluorescence micrographs of SCR, NOTCH1^KO, and ICD^KO cells labeled with phalloidin (black). Scale bar, 10 µm. (D) The intensity of cortical actin at cell–cell junctions, quantified from phalloidin-stained micrographs. n ≥ 12 fields of view from three independent experiments. (E) Traction force microscopy traction maps averaged from 10 fields of view from SCR, NOTCH1^KO, and ICD^KO cells. Scale bar, 20 µm. (F) Quantification of relative integrated monolayer tractions. n ≥ 11 traction force measurements from three independent experiments. (G) Left: Domain schematics of endogenous Notch1 and two rescue constructs utilized: Notch1 ICD overexpression (ICD O/E) and a N-terminal SNAP-tagged Notch1 TMD (SNAP-TMD). Right: Fluorescence micrographs of NOTCH1^KO cells expressing transduction control GFP (top row), SNAP-TMD (middle row), or ICD O/E (bottom row). Cells were immunostained with E-cadherin (black) and labeled with phalloidin (black). Scale bar, 20 µm. (H) Fluorescence micrograph of NOTCH1^KO cells expressing SNAP-TMD (green) and immunostained with E-cadherin (magenta). Blue arrows indicate junctions with asymmetric expression of SNAP-TMD. Scale bar, 20 µm. (I) Quantification of the frequency of focal adherens junctions in cells with symmetric expression of SNAP-TMD and asymmetric expression of SNAP-TMD. n ≥ 12 fields of view from three independent experiments. For plots B, D, and F, mean ± SEM; one-way ANOVA with Tukey’s post-hoc test, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns denotes non-significant. For plot I, mean ± SEM; two-tailed unpaired t test, **P < 0.01.