Adhesive Interactions To assess the influence of mDia1 depletion on cadherin accumulation at AJs, we measured the fluorescence intensities of E-cadherin at cell-cell contacts on immunofluorescence images (Figure 9C). We found that the fluorescence intensity of E- cadherin in IAR-2 cells transfected with mDia1 siRNA was reduced on average by ,50% in comparison with that of the control. In contrast, we did not find significant differences between the fluorescence intensities of cadherins at cell-cell contacts in control and N17Rac-loaded IAR-6-1 and Rat-1 cells. Thus, mDia1 contributes to the formation of tangential AJs in nontransformed IAR-2 epithelial cells but is not involved in the assembly and maintenance of radial AJs in transformed epithelial cells and fibroblasts. Effect of N17Rac loading on the formation of AJs Rac signaling has been shown to play an essential role in the establishment of epithelial AJs [8,9]. We also examined a possible alteration of its contribution to the regulation of AJ assembly in transformed epithelial cells. A dominant-negative form of Rac, N17Rac, was loaded into cells during the wounding of monolayers to suppress the activity of cellular Rac (Figure 10A). IAR-2 cells loaded with N17Rac did not form continuous tangential AJs. In these cells, E-cadherin could accumulate only in dot-like aggregates or short linear fragments at cell-cell boundaries. In contrast, N17Rac did not affect the formation of radial E- cadherin-based AJs in transformed IAR-6-1 cells and of N- cadherin AJs in Rat-1 fibroblasts. To assess the influence of N17Rac loading on cadherin accumulation at AJs, we also measured the fluorescence intensities of E-cadherin at cell-cell contacts in IAR-2, IAR-6-1, and N- cadherin in Rat-1 cells (Figure 10B). Analysis revealed that the fluorescence intensity of E-cadherin in N17Rac-loaded IAR-2 cells was reduced on average by ,50% in comparison with that of the control. In contrast, we did not find significant differences between the fluorescence intensities of cadherins at cell-cell contacts in control and N17Rac-loaded IAR-6-1 and Rat-1 cells. Thus, the Figure 10. Effect of N17Rac on AJ formation in different cell types. Cell monolayers were wounded in the presence of N17Rac and TRITC- labeled dextran as a marker. (A) IAR-2 cells loaded with N17Rac fail to form continuous tangential AJs (arrowhead). Inhibition of Rac activity does not affect the formation of radial AJs. IAR-6-1 cells loaded with N17Rac assemble radial AJs (arrowhead). Rat-1 fibroblasts loaded with N17Rac also form radial AJs (arrowhead). (B) Effect of N17Rac loading on cadherin accumulation at cell-cell contacts. Fluorescence intensity of E-cadherin or N-cadherin at cell-cell contacts of N17Rac-loaded cells relative to fluorescence intensity at cell-cell contacts of control cells is shown. Asterisk indicates the value that differs significantly from corresponding control (t-test, p,0.001, n = 27 contacts). Bar, 10 mm. doi:10.1371/journal.pone.0008027.g010 PLoS ONE | www.plosone.org 12 November 2009 | Volume 4 | Issue 11 | e8027