RhoJ knock-down blocks EC lumen formation. (A-B) The ability of RhoJ siRNA to repress RhoJ expression. HUVECs were transfected with various RhoJ siRNAs (siRNAs 1-4; 40 nmol/L) or a control siRNA (CTR). After incubation for 48 hours, RhoJ expression was assessed by QPCR using RhoJ-specific primers (A) or by Western blot analysis using anti-RhoJ antibody (B). Tubulin was used as a loading control. Quantitation was done by densitometric analysis (n = 3). *P < .05. (C) ECs were treated with RhoJ siRNAs 2 and 3 (40 nmol/L). After incubation for 24 hours, cells were seeded as single cells in 3D collagen type I matrices for 24 hours before preparation of fixation, staining, and photography. EC lumenal areas were quantitated using Metamorph software. Data shown are presented as values of normalized lumen area per high powered field relative to control samples ± SD, n = 20, *P < .01. Representative images of both lower- (top) and higher-powered (bottom) from lumen formation in siRNA-treated ECs are shown. Arrows indicate EC lumens. (D) RhoJ (siRNAs 2 and 3) or control siRNA (40 nmol/L) transfected HUVECs were seeded in 3D collagen matrices in the presence of pericytes to undergo tube morphogenesis as described above. Green colored cells are pericytes. Quantitation of relative vessel area was done on RhoJ 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. (E) Temporal analysis of EC tubulogenesis following siRNA suppression of ERG and RhoJ. ECs were first treated with control (CTR), ERG siRNA 4 and RhoJ siRNA 3 (40 nmol/L). After 24 hours of incubation, the lumen area was quantitated over 24 hours from time-lapse videos (see supplemental Videos 1-4). Data are shown as average EC lumenal area per high powered field relative to control ± SD, n = 5, P < .01.