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Blood, Vol. 111, Issue 4, 2015-2023, February 15, 2008
 B-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis
Blood Dimberg et al.
111: 2015
Supplemental material for: Dimberg et al
Files in this Data Supplement:
- Figure S1. αB-crystallin is up-regulated and phosphorylated in BCE cells during in vitro angiogenesis (PDF, 407 KB) -
Western blot analyses of  B-crystallin expression and the level of Ser59 phosphorylated B-crystallin in whole cell lysates of BCE cell before seeding (ps), cultured in monolayers on gelatin (proliferating cells) or 3-D collagen cultures forming tubular structures in the presence or absence of 10 ng/ml FGF-2.  -catenin expression was used as a loading control.
- Figure S2 A-B. αB-crystallin is co-localized with vimentin in proliferating endothelial cells, and translocates to granular structures during tubular morphogenesis (PDF, 580 KB) -
BCE cells proliferating on gelatin (A) or forming tubular structures in a collagen matrix (B) for 24 hours were analyzed by immunofluorescence to visualize subcellular localization of cytoskeletal components and B-crystallin, as indicated. Arrows show B-crystallin containing granular structures.
- Figure S2 C-D. αB-crystallin is co-localized with vimentin in proliferating endothelial cells, and translocates to granular structures during tubular morphogenesis (PDF, 102 KB) -
Whole cell lysates were prepared from BCE cells forming tubular structures on collagen or proliferating on gelatin for the indicated time periods. Complex formation between B-crystallin and vimentin was analyzed by immunoprecipitation of B-crystallin (using anti- B-crystallin SPA-223, Stressgen) followed by western blot using antibodies directed toward vimentin (DAKO) or B-crystallin (SPA-222, Stressgen). Lysate volumes were adjusted so that approximately equal levels of B-crystallin were immunoprecipitated for each time point. D: Immunofluorescent analysis of BCE cells forming tubular structures on collagen for 12 hours show a partial co-localization of B-crystallin and vimentin in cellular extensions. (A: Bar; 100 µm, B,D: Bar; 20 µm).
- Figure S3. Matrigel plugs from Cryab -/- mice are less vascularized than plugs from wild type mice (PDF, 635) -
To study angiogenesis in matrigel plugs in vivo, 350 µl matrigel (high concentration, BD Biosciences) containing 0.5 µg/ml VEGF A (Pepro Tech) and 0.5 µg/ml FGF-2 (PeproTech) was injected subcutaneously on the ventral side of Cryab-/- or 129SvJ wt mice; one vehicle-containing plug on one side of the mid-line and one growth factor-containing plug on the contralateral side. Matrigel plugs were harvested day 7 and frozen in isopentane/dry ice. Twenty µm sections across the entire plug were stained with antibodies against mouse CD31 (BD, Pharmingen), followed by Alexa Fluor 568-conjugated secondary antibodies (Molecular Probes). The CD31-positive vessel area within the plug was quantified using Easy Image analysis software, and normalized to the area of the plug. Left panel: Immunofluorescent staining of representative plugs from wild type and Cryab -/- mice. The white line shows the borders of the plug; only vessels inside the plugs were counted (bar; 1 mm). Right panel: The mean vessel area of multiple plugs (12 wild type and 13 Cryab-/- plugs) was quantified by Easy Image Analysis (N=12-13, mean±SD, *P<0.05).
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