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. 2016 Mar 7;126(4):1251–1266. doi: 10.1172/JCI83427

Figure 2. Increased doppel expression increases tumoral angiogenesis and EC function.

Figure 2

(A) Total volume of blood vessels in squamous, lung, breast, and colon tumor. An aliquot of tumor (1 g) was dissected to make single cells from the site where the CD31-positive area was calculated (n = 3 sections from each tumor). (B) Doppel expression in individual TECs as determined by flow cytometric analysis (3 experiments). (C) Experimental procedure for evaluation of the gain-of-function effect of doppel in ECs. Luciferase-expressing HUVECs (Hu+luc) and doppel-transfected Hu+luc (Hu+luc+dpl) spheroids were implanted s.c. in a Matrigel-fibrin matrix into female SCID mice. Three weeks after transplantation, the vascularization was analyzed. (D) Noninvasive monitoring of vascularization by bioluminescence imaging (n = 4 mice). (E) Ex vivo bioluminescence counts. ***P < 0.001 versus Hu+luc, Student’s t test. (F) Hemoglobin content within Hu+luc and Hu+luc+dpl plugs was quantified. ***P < 0.001 versus Hu+luc, Student’s t test. (G) 3D structure of the vascular network formed by Hu+luc and Hu+luc+dpl cells, as assessed by confocal microscopy using IF whole-mount staining for hCD34. Scale bar: 50 μm. (H) Immunoperoxidase detection of hCD34-positive blood vessels in Hu+luc and Hu+luc+dpl plugs. Scale bar: 20 μm. (I) Characterization and images of vascular network by staining for doppel (red), hCD34 (green), and nuclei (blue) in Hu+luc and Hu+luc+dpl plugs. Scale bar: 20 μm. (J) Quantification of hCD34-positive and doppel-positive mean vessel density (MVD) in Hu+luc and Hu+luc+dpl plugs. Doppel-positive vessels were not detected in Hu+luc plugs. ***P < 0.001 versus Hu+luc, Student’s t test. n = 4 plugs per experiment.