Adhesive Interactions Figure 9. Effect of mDia1 depletion on AJ formation. Subconfluent cultures were transfected with mDia1 siRNA or control GFP siRNA and incubated for 48 h. (A, B) Immunoblot analysis of cell lysates. Total lysate protein (20 mg) was separated with SDS-PAGE and immunoblotted for mDia1 (a-tubulin was used as a loading control). Immunoblotting (A) shows the extent of mDia1 suppression by RNAi in different cell lines. Samples 1, 2, and 3 represent GFP siRNA-transfected cells, control cells, and mDia1 siRNA-transfected cells, respectively. Densitometry (B) shows the significant reduction of mDia1 after RNAi application (OD, optical density). (C) Effect of mDia1 suppression on cadherin accumulation at cell-cell contacts. Fluorescence intensity at cell-cell contacts of mDia1 siRNA-transfected cells relative to fluorescence intensity at cell-cell contacts of GFP siRNA-transfected cells on immunofluorescence images is shown. Asterisk indicates the value that differs significantly from corresponding control (t-test, p,0.001, n = 25 contacts). (D) Effect of mDia1 suppression on the formation of AJs in a narrow wound. Cell monolayers were wounded, fixed 4 h later, and stained for E-cadherin and mDia1 simultaneously. Arrows show the newly formed AJs in control cells. The staining for mDia1 shows a significantly lower level of mDia1 expression in the cells transfected with mDia1 siRNA than in control cells. IAR-2 cells transfected with mDia1 siRNA fail to form tangential AJs (arrowhead). mDia1 is not required for the formation of radial AJs either in transformed IAR-6-1 cells or in Rat-E fibroblasts (Rat-1 fibroblasts stably expressing E-cadherin). Radial AJs (arrowheads) are formed at the sites of contacts between mDia1 siRNA-transfected cells. Bar, 10 mm. doi:10.1371/journal.pone.0008027.g009 Depletion of mDia1 did not influence the assembly of radial AJs in Rat-1 fibroblasts. To demonstrate this, we established Rat-1 cells stably expressing E-cadherin that was colocalized with N-cadherin in AJs (data not shown). Using anti-E-cadherin antibodies to localize AJs in Rat-1 fibroblasts and anti-mDia1 antibodies to reveal the cells transfected with mDia1 siRNA, we found that mDia1 suppression by RNAi did not prevent the formation of radial AJs in Rat-1 fibroblasts. PLoS ONE | www.plosone.org 11 November 2009 | Volume 4 | Issue 11 | e8027