Tests for the role of myosin II activation in migration of αEcat KO DLD1 cells and summary of the results. (A) Stable lines of αEcat KO DLD1 cells transfected with the indicated GFP-tagged MLC2 mutants were immunostained for GFP and ZO-1. Staining of nuclei is due to nonspecific binding of antibodies. (B) Myosin IIA or IIB was deleted in αE-cat KO DLD1 cells, as shown in Fig. S1 C. Cells were immunostained for ZO-1 and actin. Graphs, migration distance of cells at the wound edges 24 h after scratching of the culture, in both A and B. Data were analyzed as explained in the legend of Fig. 1 B. **, P < 0.01; ***, P < 0.001 (t test, one-sided). (C) Illustrated summary of the results. In wild-type cells, WRC and Arp2/3 components are anchored to AJ and are involved in c-lamellipodia formation when activated by undefined mechanisms (dotted arrow). In the open junction of αEcat KO cells, WRC and Arp2/3 components appear to be constitutively active, without having any specific anchoring structures. Contraction of AJ-associated actomyosin cables (two-way arrows) causes AJ disruption in αEcat KO cells. Error bars represent SEM. Scale bars, 10 µm.