ERG knock-down blocks EC lumen and tube formation in 3D collagen matrices. ECs were treated with single siRNAs targeting ERG (siRNAs 2 and 4; 40 nmol/L) or control (40 nmol/L) and incubated for 24 hours. (A) Transfected HUVECs were seeded as single cells in 3D collagen type I matrices (at 2 × 106 cells/mL) to undergo lumen formation for 24 hours before preparation of fixation, staining, and photography. Representative fields of siRNA-treated ECs from lumen formation are shown as both lower- (top) and higher-powered (bottom) images. Arrows indicate EC lumens. EC luminal areas were quantitated using Metamorph software. Data shown are presented as normalized values of lumen area per high powered field relative to control (CTR) treatment ± SD, n = 20, P < .01. (B) Transfected HUVECs were seeded in 3D collagen matrices in the presence (EC/pericyte) or absence (EC only) of pericytes to undergo tube morphogenesis for 72 hours. Quantitation of relative vessel area was done on ERG suppression versus control conditions. Data shown are presented as normalized values of lumen area per high powered field relative to control treatment ± SD, n = 20, P < .01.
