Adhesive Interactions Figure 8. Effect of myosin II inactivation on AJ formation. Cell monolayers were wounded and transferred to the medium with the Rho-kinase inhibitor Y-27632 or with the myosin II ATPase inhibitor blebbistatin for 2 h. Cells were fixed and stained for F-actin, E-cadherin, or N-cadherin. (A) Incubation with Y-27632 (30 mM) leads to total disappearance of actin bundles. In IAR-2 cells, both marginal (arrows) and straight actin bundles disappear. IAR-2 cells can form wave-like tangential AJs (arrowhead) in the presence of 30 mM Y-27632. IAR-6-1 cells treated with 30 mM Y-27632 do not assemble radial AJs. E-cadherin-based junctions are seen as dots at the cell-cell boundary (arrowhead). Y-27632 (30 mM) blocks the formation of radial AJs in Rat-1 fibroblasts (arrowhead). Blebbistatin abolishes actin bundles in all types of cells. IAR-2 cells in the presence of 50 mM blebbistatin can accumulate E-cadherin in wave-like tangential AJs (arrowhead). Myosin II activity is necessary for the assembly of radial AJs. IAR-6-1 cells treated with 50 mM blebbistatin accumulate E-cadherin in dot-like clusters (arrowhead) and do not form radial AJs. In Rat-1 fibroblasts, blebbistatin (15 mM) inhibits the formation of radial AJs (arrowhead). (B) Accumulation of E-cadherin and N-cadherin at cell-cell contacts was quantified by measuring fluorescence intensity on immunofluorescence images. Fluorescence intensity at cell-cell contacts of drug-treated cells relative to fluorescence intensity at cell-cell contacts of control cells is shown. Vertical lines indicate SEM. Asterisks indicate the values that differ significantly from corresponding controls (t-test, p,0.001, n = 25–32 contacts). Bar, 10 mm. doi:10.1371/journal.pone.0008027.g008 PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e8027