CYLD regulates angiogenesis by mediating cell spreading and migration. (A) HUVECs transfected with pEGFPC1 and pSUPER or pSUPER-CYLD plasmids for 72 hours were plated onto matrigel, and photographs were taken 25 minutes later. White arrowheads indicate cells spreading on the plates, and black arrowheads indicate cells without such a property. Objective lens used was A-Plan 20×/0.3 NA dry (Carl Zeiss Inc). (B) Experiments were performed as in panel A, and the degree of cell spreading was quantified 25 and 60 minutes after plating. Data are the mean and SE from 2 experiments (600 transfected cells were measured for each experiment; **P < .01 vs control). (C) HUVECs transfected with CYLD or control siRNAs for 72 hours were scratched, and wound margins were imaged 12 hours later. Objective lens used was A-Plan 20×/0.3 NA dry (Carl Zeiss Inc). (D) Experiments were performed as in panel C, and the extent of wound closure was quantified by measuring the wound area compared with the initial wound area. Data are the mean and SE from 2 experiments with each performed in triplicate (**P < .01 vs control). (E) HUVECs were transfected with pEGFPC1 and pSUPER or pSUPER-CYLD plasmids, and the fluorescence of GFP at the leading edge of cells was recorded at 20-second intervals with the use of the TCS SP5 confocal microscope (Leica), equipped with a live-cell imaging workstation and the LAS AF software. Rectangular regions were selected as indicated to analyze membrane ruffle dynamics. Objective lens used was HCX Plan-Apochromat 20×/0.7 NA dry (Leica). (F) Experiments were performed as in panel E, and membrane ruffle dynamics were presented as 3-dimensional surface plots.