Fig. 1. Effect of Dia1 knockdown on the integrity of adherens junctions in MCF7 cells. (A) Distribution of endogenous hDia1 in MCF7 cells, as revealed by antibody staining. Note some enrichment at cell-cell contacts. (B) -catenin and (C) actin stainings of the same field. (D) Western blot of MCF7 cells transfected with a GFP expression vector and either pSuper vector (cont; lane 1) or a shRNA sequence for human Dia1 cloned in a pSuper vector (si-hDia1; lane 2). GFP-expressing cells were collected by FACS sorting 72 hours after transfection. The blot was probed with antibody against Dia1, and an antibody against tubulin as a loading control. (E,F) Depletion of Dia1 by interfering RNA. MCF7 cells co-transfected with pGFP-C1 and pSuper si-hDia1 were stained with antibody against Dia1 72 hours following transfection. Dia1 is reduced dramatically in cells coexpressing the GFP marker (F, asterisks). (G-L) E-cadherin localization in control and si-hDia1-expressing cells. E-cadherin staining in control cells (H) and hDia1-knockdown cells (K) shows that the localization of E-cadherin at cell-cell borders is dramatically reduced in Dia1-knockdown cells (arrows). GFP labeling of transfected cells is shown in (G,J) and actin staining is shown in (I,L). White boxes in the images H and K are used for quantification (see Fig. 3C,D). Bars, 10 µm. (M) Assessment of intact E-cadherin-containing cell-cell contacts in control and hDia1-knockdown cells. The mean percentage of intact E-cadherin-positive contacts is shown by bars. Quantification was done for three independent experiments, including measurements of 30 pairs of cells for each experiment. Error bars represent standard deviations (s.d.).