JCS014365 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Comparison of the effect of Dia1 knockdown on E-cadherin and cingulin. (A-F) Confocal images of cells transfected with a vector encoding GFP together with either empty pSuper vector (A-C) or pSuper si-hDia1 (D-F) stained with antibodies to E-cadherin (B,E) and cingulin (C,F). YZ projections on the right of the corresponding images were taken along the red lines. Junctions between cells are indicated by arrows. GFP fluorescence indicates the transfected cells (A,D). Bar, 10 μm.

• Supplemental Figure S2 -

  Fig. S2. De novo formation of cell-cell contacts following a Ca²⁺ switch is impaired in Dia1-knockdown cells. (A) MCF7 cells transfected with a vector encoding GFP together with either empty pSuper (GFP+pSuper) or pSuper si-hDia1 (GFP+si-hDia1) and assayed 48 hours following transfection. The ability of the cells to re-form contacts was assessed by Ca²⁺ switch. Cells were fixed and stained for E-cadherin, cingulin and phalloidin immediately after Ca²⁺ depletion (time 0) and at 15-120 min intervals after restoration of the extracellular Ca²⁺ and compared with cells maintained in the continuous presence of Ca²⁺ (untreated). The GFP fluorescence panels indicate the transfected cells. Arrows indicate contacts between transfected cells. (B,C) Assessment of intact E-cadherin- or cingulin-containing cell-cell contacts in control (B) and hDia1-knockdown (C) cells. A quantification of the percentage of intact contacts at various stages of Ca²⁺ manipulation was performed as in Figs 1 and 2.

• Supplemental Figure S3 -

  Fig. S3. Effect of constitutively active Rho on cell-cell junctions in control and Dia1-knockdown cells. (A-C) Transfection with vector encoding GFP-RhoA-V14 leads to formation of stress fibers (B) and slightly enhances cell-cell contacts (C). (D-F) Co-transfection of GFP-RhoA-V14 together with pSuper si-hDia enhances formation of actin cables (E), but does not rescue the cell-cell junctions, as revealed by β-catenin staining (F). GFP fluorescence indicates the transfected cells. Arrows indicate cell-cell junctions. Bar, 10 μm. (G) Assessment of intact β-catenin-positive cell-cell contacts between cells expressing GFP-RhoA-V14 and GFP-RhoA-V14 together with the pSuper-si-hDia1 plasmid in comparison with hDia1-knockdown cells. Bars show the mean percentage of intact β-catenin-positive contacts. Quantification was done for three independent experiments, including measurements of 20 pair of cells for each experiment. Error bars represent standard deviations (s.d.).