Myosin light chain localization and activation in SCRIB KO cells. (A) Parental Caco-2 cells stably expressing myosin light chain MYL12A tagged with GFP and filter differentiated for 21 d were analyzed by confocal microscopy. Apical plane (left), basal plane (right), and Z-transversal sections (bottom) are represented. (B) SCRIB KO Caco-2 cells stably expressing myosin light chain MYL12A tagged with GFP and filter-differentiated for 21 d were analyzed by live confocal microscopy. Apical plane (left), basal plane (right), and transversal sections (bottom) are represented. The white arrowhead indicates basal myosin accumulation. (C) Filter-differentiated parental Caco-2 cells (top) and SCRIB KO (bottom) stained with ZO1 (left) and anti-Phospho S19 activated myosin light chain (apical plan, center panel, and basal plan, right panel). (D) Fluorescence intensity quantification of Myosin S19 (red) and Dapi (blue) along the Z axis of filter-differentiated parental Caco-2 cells. Error bars correspond to standard deviations. The graph is the average of 10 measurements. Apical and basal myosin S19 fluorescence intensity maxima are indicated. (E) Fluorescence intensity quantification of Myosin S19 (red) and Dapi (blue) along the Z axis of filter-differentiated SCRIB KO cells. Error bars correspond to standard deviations. The graph is the average of 10 measurements. (F) Basal/apical relative fluorescence myosin S19 ratio of parental and SCRIB KO cells. The P value represents the result of an unpaired two-tailed t test done on the mean of four independent experiments (red, pink, yellow, and red), each of them being the result of 10 measurements (Parental Caco-2 means: 0.9644, 0.6647, 0.5191, and 0.3406; SCRIB KO means: 1.1540, 1.037, 1.007, and 0.8906). (G) Immunostaining with anti-ZO1 (left) and anti-SHROOM2 of parental Caco-2 (top) and SCRIB KO cells (bottom). (H) Quantification of SHROOM2 intensity picks at cell–cell junctions. The results have been obtained on 3 × 15 cells. Bar is 20 µm.