JCS02660 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Relative proliferation and apoptosis rates of M2, A7, M2-CC1 and A7-CC1 cells. (A) Mean apoptosis rates and mean proliferation relative to M2 cells and their standard deviation for triplicates of typical experiments (n=3) are shown. (B) Mean numbers of cells exhibiting BrdU incorporation in an area of 50 mm² adjacent to the cleared area 24 hours after wounding are shown (n=3). Method: Cell proliferation of M2, A7, M2-CC1 and A7-CC1 was determined using the BrdU Cell Proliferation ELISA (Roche Diagnostics GmbH, Mannheim, Germany). To test spontaneous apoptosis rates, cells were incubated with FITC-conjugated annexin V antibody (Bender MedSystem, CA, USA) and 10 mg/ml propidium iodide, and were subjected to flow cytometric analysis. Fig. S1B: 24 hours after monolayer wounding, cells were incubated with 10 mM BrdU for 1 hour. Cells were fixed and permeabilized as described above and incubated for 2 hours with a 1:10 dilution of FastImmune anti-BrdU-FITC antibody/DNase (BD Biosciences, CA, USA). Samples were mounted and visualized as described above. Cells exhibiting BrdU incorporation were counted in five areas of 50 mm² adjacent to the cleared area (n=3).

• Supplemental Figure 2 -

  Fig. S2. Cell-matrix adhesion. M2, A7, M2-CC1 and A7-CC1 cells were allowed to adhere on collagen-I, collagen-IV, vitronectin, laminin-1, fibronectin, or Matrigel for 10, 30, or 60 minutes. Mean adhesion relative to M2 wild type cells and standard deviation for triplicates of a typical experiment (n=3) are shown. Method: Relative adhesion of M2, A7, M2-CC1 and A7-CC1 cells on different matrices. 96-well plates were coated overnight with 20 mg/ml fibronectin, 20 mg/ml collagen-I, 20 mg/ml collagen-IV, 20 mg/ml Matrigel (BD Biosciences, CA, USA), 10 mg/ml laminin-1 (Roche, Mannheim, Germany), or 2 mg/ml vitronectin in PBS, respectively. Unspecific binding was blocked with 1% BSA in PBS for 4 hours at RT. Cells were serum starved for 1 hour, plated in 200 ml serum-free MEM-medium (310³ cells per well) and incubated for 10, 30 or 60 minutes at 37°C. Unattached cells were removed by washing with PBS. Attached cells were fixed with 4% PFA/PBS and stained with 0.1% crystal violet. Quantification was done in a microplate reader at 570 nm after dye solubilization with Triton X-100. Adhesion of M2 cells was set to 100%. Adhesion compared with M2 cells of triplicates of one representative experiment out of two performed is shown.

• Supplemental Figure 3 -

  Fig. S3. Expression of N-cadherin and E-cadherin. (A) Indirect immunofluorescence of E-cadherin with the mAb HECD1 (left panel) and N-cadherin with the mAb #32, BD/Transduction Laboratories, CA, USA (right panel) in fixed and permeabilized M2, A7, M2-CC1 and A7-CC1 cells. (B) Confocal images of E-cadherin (CY2, green) / phalloidin-TRITC (red) stained cells. Bar, 5 µm.

• Movie 1 -

  Movie 1. For time-lapse studies of wound healing assays, monolayers of M2 cells were wounded 24 hours after plating by scratching with a pipette tip. Debris was removed by washing. Wounded monolayers were subjected immediately to time-lapse studies for 3 hours at 103 magnification using a Zeiss Axiovert 200 microscope equipped with the AxioCam digital system. The arrow indicates the area shown in detail in Fig. 5C.

• Movie 2 -

  Movie 2. For time-lapse studies of wound healing assays, monolayers of A7 cells were wounded 24 hours after plating by scratching with a pipette tip. Debris was removed by washing. Wounded monolayers were subjected immediately to time-lapse studies for 3 hours at 103 magnification using a Zeiss Axiovert 200 microscope equipped with the AxioCam digital system. The arrow indicates the area shown in detail in Fig. 5C.

• Movie 3 -

  Movie 3. For time-lapse studies of wound healing assays, monolayers of M2-CC1 cells were wounded 24 hours after plating by scratching with a pipette tip. Debris was removed by washing. Wounded monolayers were subjected immediately to time-lapse studies for 3 hours at 103 magnification using a Zeiss Axiovert 200 microscope equipped with the AxioCam digital system. The arrow indicates the area shown in detail in Fig. 5C.

• Movie 4 -

  Movie 4. For time-lapse studies of wound healing assays, monolayers of A7-CC1 cells were wounded 24 hours after plating by scratching with a pipette tip. Debris was removed by washing. Wounded monolayers were subjected immediately to time-lapse studies for 3 hours at 103 magnification using a Zeiss Axiovert 200 microscope equipped with the AxioCam digital system. The arrow indicates the area shown in detail in Fig. 5C.