MASTL regulates cell spreading, but not junctional orientation, of confluent MCF10A cells. (A and B) Representative images of E-cadherin, β-catenin, F-actin (Phalloidin-Atto), and DAPI staining, and orthogonal view of the monolayer in siControl and siMASTL (48 h silencing) confluent MCF10A cultures. Images were acquired on a 3i CSU-W1 spinning disk confocal. (C) Quantification of cell area in a monolayer based on β-catenin staining (three independent experiments, with 251 [siControl], 133 [siMASTL#6], or 189 [siMASTL#11] cells in total). (D) Quantification of relative height of monolayer based on β-catenin staining (three independent experiments, 11 [siControl], 13 [siMASTL#6], or 13 [siMASTL#11] images in total). (E) Representative images of the local orientation of β-catenin–stained junctions represented by the corresponding color assigned to each specific angle of orientation, from −90° to 90°. (F) Analysis of cell–cell junction orientation in control and MASTL-silenced cells according to β-catenin staining using the OrientationJ ImageJ plugin (see Materials and methods). Frequency distributions of relative junctional orientations were then averaged over 12 images per condition, and the frequency of junction alignment was calculated across the ±30° spanning the peak. See also Table S1.