Supplementary Figure legend

Supplementary Figure 1: Effect of cad-11 on adhesion of ES cells to collagen-coated plates.

ES cell lines transfected with cad-11 shRNA (ES/shCad-11 cells) or control shRNA cells (ES/shCtr cells) were plated on collagen-coated plates and the number of attached cells was counted. Results are given as means ± SD. N.S., Not Significant.

Supplementary Figure 2: Expression of cad-11 in RD-ES/shCad-11/luc, RD-ES/shCtr/luc, TC-71/shCad-11/luc, and TC-71/shCtr/luc cells.

Expression measured by (a) Real-time PCR (means ± SD; **P < 0.001) and (b) Western blotting. (c) Proliferation rates of RD-ES/shCad-11/luc, RD-ES/shCtr/luc, TC-71/shCad-11/luc, and TC-71/shCtr/luc cells. Experiments were performed in triplicate, and data are expressed as means ± SD. (d) The intensity of bioluminescence in RD-ES/shCad-11/luc, RD-ES/shCtr/luc, TC-71/shCad-11/luc, and TC-71/shCtr/luc cells.

Supplementary Figure 3: Effect of cad-11 on homophilic adhesion of ES cells.

RD-ES/shCad-11/luc, RD-ES/shCtr/luc, TC-71/shCad-11/luc, and TC-71/shCtr/luc cells were seeded on the same cell line (RD-ES or TC-71) layer. After 24 hours cells were observed and analyzed with an IVIS 200 Imaging System (Xenogen). Relative luciferase intensities of RD-ES/shCad-11/luc, RD-ES/shCtr/luc, TC-71/shCad-11/luc, and TC-71/shCtr/luc were compared in the presence and absence of ES monolayer cells. Data are depicted as means ± SD of at least three independent experiments. N.S., Not Significant

Supplementary Figure 4: (a) Expression of other cadherins in Ewing sarcoma cell lines were measured by Western blotting. N-cadherin was expressed exclusively in RD-ES cells and E-cadherin was expressed in SK-N-MC, SK-ES-1, and TC-71 cells. Positive control were Hela (cervical epithelial carcinoma cell line) in expression of N-cadherin and H2228 (lung adenocarcinoma) in expression of E-cadherin. (b)RD-ES cells were incubated with 20μg/ml anti-N-cadherin–neutralizing antibody or IgG (20μg/ml) and SK-N-MC, SK-ES-1 and TC-71 cells were incubated with 20µg/ml anti-E-cadherin-neutralizing antibody or IgG (20μg/ml) then subjected to the adhesion assay. Data are depicted as means ± SD of at least three independent experiments (**P < 0.01 vs. control IgG).

Supplementary Figure 5: Effects of cad-11 on expression of MMP-2 and MMP-9.

For the gelatin typographic analysis, confluent RD-ES, SK-N-MC, SK-ES-1, and TC-71 cells were treated with serum-free medium for 48 hours. Concentrated conditioned medium from the ES cells was electrophoresed on a 10% polyacrylamide gel containing gelatin.Conditioned medium of HT1080 was used as a positive control.

Supplementary Figure 6: Bioluminescence image of mice 6 weeks after intracardiac injection of RD-ES/shCtr-11/luc or RD-ES/shCad-11/luc cells (a) and TC-71/shCtr-11/luc or TC-71/shCad-11/luc cells (b). (c) Necropsy data. Anatomical site of bone metastases of the mice injected with RD-ES/shCtr/luc cells (hind limb, 2; mandible, 4; spine, 2), and the mice injected with RD-ES/shCad-11/luc cells (mandible, 1; spine, 1). Anatomical site of bone metastases of the mice injected with TC-71 shCtr/luc cells (hind limb, 9; mandible, 8; spine, 9), and the mice injected with RD-ES/shCad-11/luc cells(hind limb, 2; mandible, 2; spine, 4).


Supplementary Materials and Methods

Adhesion blockage assay

The cover glasses were coated with 10 μg/ml recombinant human N-cadherin-Fc chimera (R&D Systems), 10 μg/ml E-cadherin-Fc chimera (R&D Systems), or 10 μg/ml IgG-Fc (R&D Systems) at 4°C overnight. After blocking with 1% bovine serum albumin, 5×104 cells were seeded on coated cover glasses and incubated at 37°C with 20 μg/mL IgG, 20 μg/mL mouse anti N-cadherin antibody (Sigma-Aldrich) or 20 μg/mL mouse anti E-cadherin antibody(Invitrogen). After 2 hours, cover glasses were gently washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. The cover glasses were stained with Diff-Quick stain. Slides were mounted with Vectashield mounting medium (Vector Laboratories) and examined with a microscope. The numbers of cells in six randomly chosen fields were counted.

Gelatin zymography

The cells were incubated in serum-free medium. After incubating for 48 hours, the conditioned medium was collected, clarified by centrifugation, and then concentrated. Aliquots of the concentrated, conditioned media were dissolved in SDS sample buffer in the absence of reducing agents and without boiling. The samples were electrophoresed on a 10% Zymogram (Gelatin) Gel (Invitrogen) with Tries-Glycine SDS running buffer. The gel was incubated in Zymogram Renaturing Buffer (Invitrogen) for 30 minutes and then in Zymogram Developing Buffer (Invitrogen) overnight. The gel was then stained with Coomassie Blue and destained in 45% methanol / 10% acetic acid. The gelatinolytic activities of type IV collagenases appeared as clear bands against the blue background of stained gel. The gelatinolytic activities of types 2 and 9 matrix metalloproteinases appeared as clear bands against a blue stained background.

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