Twist1 induced single cell dissemination despite membrane-localized adherens junction proteins. (A and B) E-cad deletion blocks branching and induces epithelial disorganization. (C) Rare E-cad⁻ disseminated cells maintain a rounded morphology. (D and E) Twist1 expression blocks branching and induces single cell dissemination (red arrowheads). (F) Disseminated Twist1⁺ cells exhibit extensive actin-rich protrusions (red arrows). (G) In time-lapse movies, E-cad⁻ cells were only rarely observed to disseminate. In contrast, >100 cells per Twist1⁺ organoid were routinely observed to disseminate. n, number of time-lapse movies; r, number of biological replicates. (H) By Western blot, Twist1 expression resulted in reductions in protein levels of E-cad, N-cad, αE-catenin, and β-catenin (see also Fig. S3, A and B). Whole cell lysate samples were loaded for equal protein based on BCA analysis. (I–N) Membrane-localized E-cad and β-catenin (arrowheads) were detected in basally positioned cells protruding into the ECM (I and L), in cells that had just disseminated (J and M), and in disseminated cells migrating through the ECM (K and N). Bars: (B and E) 20 µm; (C, F, and I–N) 10 µm.