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Blood, Vol. 113, Issue 19, 4810-4818, May 7, 2009
Distinctive localization and opposed roles of vasohibin-1 and vasohibin-2 in the regulation of angiogenesis
Blood Kimura et al.
113: 4810
Supplemental materials for: Kimura et al
Files in this Data Supplement:
- Figure S1. A mouse model of subcutaneous angiogenesis (JPG, 73.6 KB)
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(A) Bilateral skin incisions were made on the backs of mice. The skin and subcutaneous tissue between the incisions was exfoliated from the deeper layer, and a silicon sheet was inserted beneath the flap. (B) The area of inserted silicon was indicated on the right sided photo of day 0. Neo-vessels distributed but the central region became necrotic over the experimental period. Dashed lines on the right sided photo of day 7 indicate the necrotic edge.
- Figure S2. A mouse model of subcutaneous angiogenesis (JPG, 53.7 KB)
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(A) After 7 days, the skin flap was harvested and oriented parallel to the original incisions for sectioning. Hematoxylin and eosin staining of the skin flap was performed. The arrow indicates the necrotic edge. Scale bar is 500 µm. (B) The hypoxic region was determined by pimonidazol staining. The arrow indicates the necrotic edge. Scale bar is 100 µm.
- Figure S3. Expression of VASH2 mRNA in mice (JPG, 41.1 KB)
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RT-PCR analysis of mouse adult tissues and placenta (upper) and whole embryos at Days 7.5-12.5 (lower) was performed as described in Materials and Methods. VASH2 mRNA was detected in adult brain, placenta, and whole embryos.
- Figure S4. Endogenous VASH2 in the termination zone (JPG, 30.6 KB)
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Expression of CD31 and VASH2 in the termination zone was determined by immunostaing. Scale bars are 100 µm.
- Figure S5. In vitro effect of VASH1 and VASH2 on endothelial proliferation (JPG, 39.5 KB)
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MS1 cells were stably transfected with human VASH1 expression vector or human VASH2 expression vector. Mock transfectants and either of VASH1 or VASH2 transfectants (bulk) were plated (5 × 105), and the cell number was determined after a 3 days incubation. Data is expressed as the means and SDs. *p<0.01. The expression of VASH1 or VASH2 was determined by RT-PCR. Common primer pairs were used to detect endogenous mouse as well as transfected human VASH1 or VASH2 mRNAs.
- Figure S6. Intracellular localization of VASH1 and VASH2 (JPG, 50.6 KB)
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VASH2-NT-GFP or VASH2-CT-GFP vector was transfected into GM7373 cells. After transfection, AdVASH1 was infected and immunostaining was performed using anti-human VASH1 mAb and rhodamine-conjugated secondary Ab. Images were obtained with confocal microscopy. VASH1 (red), VASH2 (green).
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