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Blood, Vol. 110, Issue 8, 2899-2906, October 15, 2007
Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin
Blood Shi et al.
110: 2899
Supplemental materials for: Shi et al, Vol. 110, Issue 8, 2899-2906.
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 98.8 KB)
- Figure S1. Endostatin-binding proteins (PDF, 170 KB) -
(A-G) Immunoblots for integrin  5 (A), integrin  1 (B), laminin-1 (C), MMP2 (D), tropomyosin (E), glypican-1 (F), and NL (G) were performed with control fractions, membrane fractions (MF), SDS sample buffer-treated fractions (2% SDS), and 500 mM sodium chloride eluted fractions, as shown.
- Figure S2. Cell cycle positions of different cell growth states (PDF, 155 KB) -
(A-C) Cell cycle positions of proliferating cells (A), serum-starved cells (B), and serum-starved cells rescued with serum and bFGF (C) were detected by flow cytometry. (D) The expression level of cell surface NL of HMECs with their different growth states was evaluated by flow cytometric analysis. The cell surface NL was recognized by anti-NL, followed by FITC-labeled secondary antibody. FITC-labeled goat IgG was used as negative control.
- Figure S3. The co-localization among biotinylated ES, the anti-NL, CD31, and Ki67 (PDF, 490 KB) -
Biotinylated ES and anti-NL antibody were simultaneously injected intravenously into mice bearing B16/F10 tumors. Colocalizations between biotinylated ES and CD31, between anti-NL and CD31, and between anti-NL and Ki67 were observed on the blood vessels of tumor. Biotinylated ES and CD31 in tumor were recognized with TRITC-conjugated streptavidin and anti-CD31, respectively, followed by FITC-conjugated goat antimouse IgG. Anti-NL and CD31 in tumors were recognized with FITC-conjugated goat antirabbit IgG and anti-CD31, respectively, followed by TRITC-conjugated goat anti-mouse IgG. Anti-NL and Ki67 in tumors were recognized with TRITC-conjugated goat antirabbit IgG and anti-Ki67, respectively, followed by FITC-conjugated goat antimouse IgG. Arrows indicate blood vessels in the field. Scale bar, biotinylated ES and CD31, anti-NL and CD31, 20 µm; anti-NL and Ki67, 100 µm.
- Figure S4. Xenograft B16/F10 melanoma growth assay (PDF, 37 KB) -
(A,B) Saline, ES (2 mg/kg), ES (2 mg/kg) along with nonimmune rabbit IgG (2 mg/kg), nonimmune rabbit IgG (2 mg/kg), rabbit anti-NL antibody (2 mg/kg), or ES (2 mg/kg) along with rabbit anti-NL antibody (2 mg/kg) were injected intraperitoneally into B16/F10-bearing nude mice at a site remote from the inoculated tumor every other day once the tumor volume reached 100 mm3. The tumors of mice were measured every 4 days, and the volumes were calculated (A). After ten injections, the mice were killed, and the tumors were resected and weighed (B).
- Figure S5. Difference responses to endostatin (PDF, 218 KB) -
(A-H) HMECs, HUVECs, hemangioma endothelial cells (HemECs), MDA-MB-435 cells, Hela cells, Colo205 cells, LLC cells, and B16/F10 cells were incubated with ES for 1 hour. ES and the cell surface NL were recognized with anti-ES or anti-NL, followed by FITC-labeled secondary antibody. The amount of ES and cell surface NL were evaluated by flow cytometric analysis. FITC-labeled goat IgG was used as negative control. (I) HMECs, HUVECs, HemECs, MDA-MB-435 cells, Hela cells, Colo205 cells, LLC cells, and B16/F10 cells were incubated with ES for 2 hours. ES, which was internalized into the nuclei of these cells, was detected by immunoblotting with anti-ES antibody. Actin served as loading control. (J) Cell proliferation assay was performed with HMECs, HUVECs, HemECs, MDA-MB-435 cells, Hela cells, Colo205 cells, LLC cells, and B16/F10 cells. The cells were incubated with 40 µg/mL ES for 48 hours. The cell number was evaluated by MTT assay (n=4). *P<.01.
- Figure S6. The specificity of the prepared anti-NL antibody (PDF, 786 KB) -
(A-C) Whole HMEC, ECV304, and HEK293T cells were applied to SDS-PAGE, and immunoblots were performed with prepared rabbit anti-NL antibody (A), prepared murine anti-NL antibody (B), or commercial available monoclonal antibody against NL (Santa Cruz) (C). (D, E) Soluble recombinant NL was applied to compete with cell surface NL to bind the anti-NL antibody. Soluble recombinant NL was added into HMEC-cultured media 10 minutes prior to the anti-NL. The molar ratios between soluble recombinant NL and the anti-NL antibody (rabbit Rb or murine Mo) are 1:0.01, 1:1, and 1:100. After 1 hour incubation, the cell surface binding rabbit (Rb) and murine (Mo) anti-NL antibody were detected by FITC-conjugated goat antirabbit IgG and FITC-conjugated goat anti-mouse IgG, respectively. DAPI indicates the cells in the field. Soluble recombinant NL was absent in the positive control group, and anti-NL antibody was absent in the negative control. Scale bar, 20 µm.
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