Adhesive Interactions Figure 7. Rho activity is required for the assembly of tangential and radial AJs. Cell monolayers were wounded in the presence of C3 transferase and TRITC-labeled dextran (as a marker). Cells were fixed and stained for E-cadherin or N-cadherin. In control cultures, AJs are formed at the sites of cell-cell contacts (arrows). The IAR-2, IAR-6-1, and IAR1170 cells loaded with C3 are unable to assemble new AJs (arrowheads). Bar, 10 mm. doi:10.1371/journal.pone.0008027.g007 and led to cell arborization (data not shown). In the case of Rat-1, we used a lower dose of blebbistatin (15 mM), which also destroyed actin bundles and stopped the assembly of radial AJs. To quantify the effects of Y-27632 and blebbistatin on cadherin accumulation at AJs, fluorescence intensities of E-cadherin and N- cadherin at cell-cell contacts were measured as described previously [23]. Analysis of fluorescence intensity revealed that Y-27632 and blebbistatin dramatically reduced the accumulation of E-cadherin at AJs in IAR-6-1 cells and of N-cadherin at AJs in Rat-1 cells (Figure 8B). In contrast, we did not find significant differences between the average levels of E-cadherin fluorescence at cell-cell contacts in control and drug-treated IAR-2 cells. The studies performed suggest that the establishment and maintenance of radial AJs both in transformed epithelial cells and in fibroblasts depend on myosin II-mediated contractility. In contrast, myosin II is not required for the assembly of tangential AJs in nontransformed epithelial cells. Effect of depletion of mDia1 on the formation of AJs To investigate the involvement of another Rho effector, mDia1 formin, in the formation of AJs, we applied the RNAi technique for the depletion of mDia1 [29]. We used two types of mDia1 siRNA duplex that had similar effects. We assayed the efficiency of suppression of mDia1 using Western blot analysis of cell lysates 48 h post-transfection. Transfection with mDia1 siRNA signifi- cantly decreased the expression of mDia1 in all three cell types (Figure 9A and 9B). For double immunofluorescence staining of mDia1 and AJs, we used E-cadherin mouse monoclonal antibodies (IgG2a isotype) and mDia1 antibodies (IgG1 isotype) revealed by secondary mouse IgG1 and mouse IgG2a antibodies. Comparison of mDia1 immunofluorescence staining of cultures revealed a dramatic decrease of mDia1 in cells 48 h after transfection (Figure 9D). Depletion of mDia1 resulted in a prominent disturbance in the formation of tangential AJs in IAR-2 cells. In all cell pairs with diminished staining of endogenous mDia1 as a result of mDia1 suppression by RNAi, tangential AJs did not assemble. At the sites of cell-cell contacts, E-cadherin could accumulate only in dot-like clusters. These data are in agreement with the results of Carramusa et al. [23] concerning the disappearance of AJs in MCF7 epithelial cells after short-hairpin RNA knockdown of Dia1. We also compared the effects of mDia1 depletion on the formation of radial AJs in transformed IAR-6-1 epithelial cells and Rat-1 fibroblasts. Unlike tangential AJs, radial AJs assembled in mDia1 siRNA-transfected cells. Double immu- nofluorescence staining revealed that in IAR-6-1 cells transfected with mDia1 siRNA, E-cadherin accumulated in radial AJs. PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e8027