Fig. 4. Effects of WAVE2 RNAi in MDCK cells. (A) Depletion of WAVE2 in MDCK cells inhibits lamellipodium formation on collagen. CTRL or WAVE2 siRNA-treated cells were trypsinized and then plated on collagen. After 4 hours, cells were fixed and stained for WAVE2 and actin filaments (F-actin). Insets in each panel show the magnified images of the areas indicated by white squares. Bar, 10 µm. The lower graph shows the number of cells with lamellipodia. The percentage of cells with lamellipodia covering 50-100% of the cell perimeter was presented as >50%. 20-50%, 20-50% of the perimeter; <20%, 0-20% of the perimeter. Data are mean ± s.d. Three independent experiments were performed. (B) Morphologies of colonies of CTRL or WAVE2 siRNA-treated cells. Bar, 100 µm. (C,D) Ca²⁺-switch assay with subconfluent MDCK cell layers. Subconfluent CTRL siRNA (C) or WAVE2 siRNA (D) treated cell layers were treated with 5 mM EGTA for 30 minutes. Cell layers were then treated with Ca²⁺-containing medium for the indicated times. Cell layers were fixed and then stained for WAVE2 and E-cadherin. Merged images of WAVE2 (red) and E-cadherin (green) are shown in insets. Insets in each panel show the magnified images of the areas indicated by white squares. Scale bar, 15 µm. (E) Quantification of cell-cell contacts with E-cadherin staining. The number of cell-cell contacts with E-cadherin staining was counted during the Ca²⁺-switch assay in C and D. Data are mean ± s.d. ^*P<0.05. Statistical significance was examined with the Student's t-test. Three independent experiments were performed.