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Blood, Vol. 109, Issue 4, 1524-1532, February 15, 2007
Deletion of tetraspanin Cd151 results in decreased pathologic angiogenesis in vivo and in vitro
Blood Takeda et al.
109: 1524
Supplemental materials for: Takeda et al, Vol 109, Issue 4, 1524-1533
Files in this Data Supplement:
- Figure S1. CD151 staining of human tumor tissue (JPG, 126 KB)
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Cancer and matching normal tissue arrays, fixed with formalin, were from Accurate Chemicals (Westbury, NY). After paraffin removal, sections were rehydrated and placed in heated retrieval solution (DakoCytomation, Fort Collins, CO). Then they were incubated with anti–human CD151 mAb, RLM30, diluted 1:100 (Novocastra, Newcastle upon Tyne, United Kingdom) and anti–von Willebrand factor mAb (DakoCytomation) for 1 hour. Sections without primary antibody served as negative controls. The Vectastain ABC Elite system (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB; DakoCytomation) were used to produce localized staining. Panels shown are each representative of 6 normal and 6 tumor sections stained. Bar represents 100 µm.
- Figure S2. Effect of CD151 deletion on normal retinal vasculature (JPG, 56.3 KB)
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Eyes were removed from 12-week-old male mice, fixed in 10% formalin for 1 hour, and then retinas were isolated under a dissecting microscope. Retinas were preincubated with 0.5% Triton X-100 in PBS for 1 hour and then stained with FITC-lectin BS1 (Bandeiraea simplicifolia; Sigma, St Louis, MO), diluted 1:100, in blocking solution overnight at 4ºC. Superficial radial and collateral vessels are shown. There was no difference in structure and no hemorrhages observed in retinas from 4 different CD151-null and CD151 wild-type mice each.
- Figure S3. CD151 effects on neovascularization in an oxygen-induced retinopathy model (JPG, 65.8 KB)
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(A) To achieve oxygen-induced retinopathy (as described by L.E. Smith et.al., 1994, Invest. Opthalmol., 35:101-111), postnatal day 7 (P7) mice (together with nursing mother) were exposed to 75% oxygen for 5 days in a custom-built chamber. On P12, mice were returned to normoxia and killed at P17. Staining of retinal vasculature revealed no difference between CD151 WT and CD151-null mice. (B) A minimum of 6 sections from each of 3 different eyes, from WT and null mice, was stained with hematoxylin and eosin. Counting of vascular cell nuclei, present beyond the inner limiting membrane into the vitreous, revealed essentially no difference between CD151 WT and CD151-null mice. Representative sections are shown. Arrows point to new blood vessels grown beyond the inner limiting membrane. Bar represents 50 µm.
- Figure S4. CD151 minimally affects endothelial cell proliferation, adhesion, and surface antigen expression (JPG, 83.3 KB)
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(A) Proliferation data (mean ± SEM) was obtained over 3 days using the MTT colorimetric assay, which measures absorbance at 490 nm (N = 3). Results shown are representative of 3 independent experiments with similar outcomes. (B) MLECs were labeled with BCECF-AM, plated for 30 minutes on plastic surfaces (coated with FN, Col, Gel, LM-1, or Matrigel) and then adhesion was quantified as described24 (N = 3). (C) Expression of the indicated antigens on the surface of MLECs was determined by flow cytometry. Negative control peaks were obtained using secondary antibody alone. Similar results were obtained in 3 different experiments.
- Figure S5. CD151 deletion does not affect endothelial cell MMP-2 and MMP-9 (JPG, 51.5 KB)
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MLECs from WT and CD151-null mice were incubated in serum-free DMEM and in the presence or absence of PMA (10 ng/mL). Culture supernatants were examined by gelatin zymography to reveal MMP-2 (pro and active forms) and MMP-9 (pro and active forms). Supernatants from mouse embryonic fibroblasts (MEFs) were run as controls for MMP-2 and MMP-9. Similar results were obtained from 3 different experiments.
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