CYLD is critical for angiogenic response in vivo. (A left) An athymic nude mouse with a semiclosed angioreactor implanted into the dorsal flank. The asterisk indicates the site for surgical incision. (Right) A semiclosed angioreactor. The boxed area indicates the region photographed. (B) Vascular growth in the angioreactors implanted in mice for 11 days. Angioreactors filled with extracellular matrix containing angiogenic factors serve as the positive control, and angioreactors filled with extracellular matrix not containing angiogenic factors serve as the negative control. In siRNA experiments, angioreactors were filled with extracellular matrix containing angiogenic factors and CYLD or control siRNAs. Objective lens used was A-Plan 4×/0.1 NA dry (Carl Zeiss Inc). (C) Immunofluorescence microscopy of blood vessels in angioreactors. Frozen sections of the vessel-containing matrigel were stained with anti-CD31 and anti–desmin antibodies to detect endothelial cells and mural cells, respectively. Objective lens used was A-Plan 40×/0.5 NA dry (Carl Zeiss Inc). (D) Immunofluorescent images of HUVECs stained with anti-CYLD or IgG control antibodies and the DNA dye DAPI. Objective lens used was Plan-Apochromat 63×/1.25 NA oil (Carl Zeiss Inc). (E) Immunoblot analysis of CYLD, CD31, Hsp90, and MMP2 in the culture medium, plasma membrane, and lysate of HUVECs. (F) Vascular growth in the angioreactors filled with extracellular matrix containing angiogenic factors and anti-CYLD antibody or IgG control. Objective lens used was A-Plan 4×/0.1 NA dry (Carl Zeiss Inc). (G) Experiments were performed as in panels B and F, and angiogenic responses were quantified by FITC-lectin staining of cell pellets recovered from the angioreactors. Data are the mean and standard error from 2 experiments with each performed in octaplicate (*P < .05).