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Figure 2


Fig. 2. Ectopic expression of mouse Dia1 rescues the effect of human Dia1 knockdown on cell-cell junctions. (A) The human Dia1-siRNA sequence used to knockdown the endogenous Dia1 contains three nucleotide mismatches compared with the sequence of mouse Dia1 (mDia1; black ovals). (B) The expression of GFP-mDia1 full-length protein is not affected by the hDia1-siRNA sequence. Control MCF7 cells, stably expressing the shRNA against LacZ (cont; lane 1) and cells stably expressing the shRNA against hDia1 (lane 2) were transiently transfected with the GFP-mDia1 plasmid (lanes 3, 4). Western blots of whole-cell lysates show that exogenous mDia1 is expressed at the correct molecular mass in the hDia1-knockdown cells (lane 4). (C,D) Expression of pSuper si-hDia1 leads to disappearance of E-cadherin staining from the interface between transfected (GFP-labeled) cells. (E,F) In cells expressing the GFP-mDia1 protein together with the human Dia1-siRNA plasmid, E-cadherin staining is as prominent as in the neighboring control cells. Bar, 10 µm. (G) Assessment of E-cadherin-containing cell-cell contacts in control cells, in hDia1-knockdown cells and in cells expressing the GFP-mDia1 protein together with the human Dia1-siRNA plasmid. Bars show the mean percentage of intact E-cadherin-positive contacts between transfected cells; error bars represent standard deviations (s.d.).





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