JCS03311 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Effects of cytochalasin D on cell-cell adhesions. MDCK cell layers were treated with 10 μM cytochalasin D for the indicated times. Cell layers were fixed and stained for E-cadherin and actin filaments (F-actin). Bar, 15 μm.

• Supplemental Figure 2 -

  Fig. S2. Formation of tight junctions. Ca²⁺-switch assay was performed with CTRL or WAVE2 siRNA-treated subconfluent cell layers. Subconfluent cell layers were treated with 5 mM EGTA-containing medium and then incubated with calcium-containing medium for the indicated times. Cell layers were fixed and stained for occludin and WAVE2. Merged images of occludin (green) and WAVE2 (red) are shown. Inset shows the magnified image of the area indicated by a smaller square. The areas indicated by solid or broken lines indicate WAVE2-positive or -negative cell-cell adhesions, respectively. Bar, 15 μm. Graphs show the results of quantification of occludin recruitment to cell-cell contacts. Cell-cell contacts were classified into three categories on the basis of the occludin staining pattern. Categories were continuous signals positive (continuous), discontinuous signals positive (discontinuous) and no signal (negative). All data are mean ± s.d. Three independent experiments were performed.

• Supplemental Figure 3 -

  Fig. S3. Effects of EGTA treatment on cell-cell adhesions. (A) Detergent solubility of WAVE2. Confluent MDCK cell layers with or without EGTA-treatment were incubated with lysis buffer containing 0.5 or 1.0 % Triton X-100 (see Materials and methods, pull-down assay). Cell lysates were collected with a cell scraper and centrifuged at 12,000 g for 15 minutes. The supernatant and the pellet were defined as the Triton-soluble and -insoluble fractions, respectively. Levels of E-cadherin, WAVE2 and actin in the detergent-soluble (S) and -insoluble (I) fractions were measured by immunoblotting. (B) Protein complexes were immunoprecipitated from lysates of EGTA-treated confluent MDCK cell layers with anti-WAVE2 antisera. Immunoprecipitates were immunoblotted for E-cadherin and WAVE2. Negative controls for immunoprecipitations were pre-immune antisera (Pre-Immune IP). Before immunoprecipitation, cell lysates were incubated with Immunoprotein A beads (Pre Incubated beads) to prevent their non-specific binding with E-cadherin.

• Supplemental Figure 4 -

  Fig. S4. DECMA-1 (anti-E-cadherin antibody) inhibits recruitment of WAVE2 to cell-cell contacts. Trypsinized cells were plated on coverslips and treated with DECMA-1. After incubation for 12 hours, cells were fixed and stained for WAVE2 and actin filaments (F-actin). Arrows indicate the lamellipodial edge. Bar, 7.5 μm.

• Supplemental Figure 5 -

  Fig. S5. Effects of WAVE2 depletion on cell-cell adhesions. Cells treated with the indicated siRNAs were fixed and stained for WAVE2, E-cadherin and actin filaments (F-actin). In addition to WAVE2 RNAi used through this manuscript WAVE2 (348), two different siRNAs for WAVE2 were examined WAVE2 (55) and (420). The areas indicated by solid or broken lines show WAVE2-positive or -negative cell-cell adhesions, respectively. Left and right insets in each image show the magnified images of WAVE2-positive or -negative cell-cell adhesions, respectively. Bar, 15 μm. (B) Cells treated with siRNAs against CTRL and WAVE2 (55, 348 and 420) were lysed, and the amount of WAVE2 examined by immunoblotting. The results of two independent experiments are shown.

• Supplemental Figure 6 -

  Fig. S6. Expression of WAVE2-GFP rescued the defects of cell-cell adhesions caused by WAVE2 RNAi. GFP or WAVE2-GFP expressing vectors were co-transfected with siRNA for WAVE2. (A) Cells were fixed and stained for β-catenin, GFP and actin filaments (F-actin). The areas indicated by solid or broken lines indicate cell-cell adhesions between GFP-negative or -positive cells, respectively. Bar, 15 μm. (B) Signal intensities of β-catenin and actin filaments were quantified with ImageJ software. Quantification was performed with cell-cell adhesions between GFP-negative or -positive cells in the same image. All data are mean ± s.d. *P<0.0001. Statistical significance was examined with Student’s t-test.

• Supplemental Figure 7 -

  Fig. S7. Inhibition of VASP/Mena functions in MDCK cells. TD-GFP, a dominant-negative mutant of VASP, was expressed in non-treated or Arp2 siRNA-treated MDCK cells. (A) Expression of TD-GFP in MDCK cells. Anti-GFP western blot is shown. (B) GFP or TD-GFP expression vectors were transfected or co-transfected with siRNA for Arp2. Transfected cell layers were fixed and stained for β-catenin, GFP and actin filaments (F-actin). The areas indicated by solid or broken lines indicate cell-cell adhesions between GFP-negative or -positive cells, respectively. Bar, 15 μm. (C) Signal intensities of β-catenin and actin filaments were quantified with ImageJ software. Formin indicates formin-1 RNAi treatment (see Fig. S7). Quantification was performed with cell-cell adhesions between GFP-negative or -positive cells in the same image. All data are mean ± s.d. *P<0.001; **P<0.0001. Statistical significance was examined with Student’s t-test.

• Supplemental Figure 8 -

  Fig. S8. Depletion of formin-1 in MDCK cells. (A) The effect of formin-1 RNAi was analyzed by RT-PCR. (B) Control siRNA, siRNAs for formin-1 or Arp2 or mixed siRNA (formin-1 and Arp2) were co-transfected with GFP expression vectors. Transfected cell layers were fixed and stained for β-catenin, GFP and actin filaments (F-actin). The areas indicated by solid or broken lines indicate cell-cell adhesions between GFP-negative or -positive cells, respectively. Bar, 15 μm. Signal intensities of β-catenin and actin filaments were quantified and shown in Fig. S6.

• Supplemental Figure 9 -

  Fig. S9. Ca²⁺-switch assay with subconfluent MDCK cell layers. Subconfluent CTRL siRNA- or WAVE2 siRNA-treated cell layers were treated with 5 mM EGTA-containing medium 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, E-cadherin, and actin filaments (F-actin). The images at the three different phases (basal, junctional and apical) are shown. The areas indicated by solid or broken lines show WAVE2-positive or -negative cell-cell adhesions, respectively. Bar, 15 μm.

• Supplemental Figure 10 -

  Fig. S10. Effects of WAVE2 RNAi on pre-adhesive structures. CTRL or WAVE2 siRNA-treated cells were plated on collagen-coated coverslips and then incubated for 2 hours. Cell layers were treated with 5 mM EGTA-containing medium and then incubated with Ca²⁺-containing medium for the indicated times. Cells were fixed and stained for WAVE2, E-cadherin and actin filaments (F-actin). Insets show the magnified images of the areas indicated by white squares. Bars, 15 μm.

• Supplemental Figure 11 -

  Fig. S11. Maturation of cell-cell adhesions. Ca²⁺-switch assay was performed with CTRL or WAVE2 siRNA- treated confluent cell layers. Confluent cell layers were treated with 4 mM EGTA and then incubated with Ca²⁺-containing medium for the indicated times. Cell layers were fixed and stained for WAVE2, β-catenin and actin filaments (F-actin). The areas indicated by solid or broken lines show WAVE2-positive or negative cell-cell adhesions, respectively. Bar, 15 μm.

• Supplemental Figure 12 -

  Fig. S12. Effects of cytochalasin D on cell-cell adhesion formation. Ca²⁺-switch assay was performed with DMSO or cytochalasin-D-treated confluent cell layers. (A) Cytochalasin D treatment before re-addition of Ca²⁺. Confluent cell layers were treated with 4 mM EGTA (+EGTA) for 30 minutes and then incubated in DMSO- or cytochalasin-D-containing medium with EGTA for 30 minutes. Such cell layers were further incubated in medium containing DMSO (DMEM + DMSO) or cytochalasin D (DMEM + Cyto D) without EGTA for 60 minutes. Cell layers were fixed and stained for β-catenin and actin filaments (F-actin). Bar, 15 μm. (B) Cyotochalasin D treatment after re-addition of Ca²⁺. Confluent cell layers were treated with 4 mM EGTA and then incubated with Ca²⁺-containing medium for 30 minutes (+ DMEM). Cell layers were further incubated in DMSO (+ DMSO) or cytochalasin D (+ Cyto D) medium for the indicated times. Cell layers were fixed and stained for β-catenin and actin filaments (F-actin). Bar, 15 μm. Timetables of the experiments (A and B) are shown. Numbers indicate the timing of fixation of specimen shown in A and B.

• Supplemental Figure 13 -

  Fig. S13. Effects of dominant-negative WAVE2 expression on cell-cell adhesions. MDCK cells stably expressing dominant-negative WAVE2 were established. Results of experiments with two independent lines (DN-14 and -16) were shown. (A) Expression of FLAG-tagged-WAVE2DN. In the presence of doxycycline (Dox), expression of the FLAG-tagged WAVE2DN was inhibited. (B) Cell layers cultured in the presence or absence of Dox were fixed and stained for β-catenin and actin filaments (F-actin). Bar, 15 μm.