Reexpression of wild-type EB1 restores vascular tube formation in EB1 siRNA–treated ECs, but a C-terminal mutant of EB1 without affinity for p150^Glued or Clasp1 fails to rescue this phenotype. (A) EB1 siRNA–treated cells were infected with adenoviral vectors carrying GFP or EB1 and allowed to undergo EC tube morphogenesis for 24 hours. Reexpression of EB1 (but not control GFP expression) successfully restores vascular tube formation in these siRNA-treated ECs. Data are presented as lumen area ± standard deviation (SD; n = 6), which was measured by Metamorph software. Statistical significance ***P < .0005. (B) Cell lysates were prepared from the samples in (A) and immunoblots were performed with the indicated antibodies. siRNA suppression of EB1 decreased both acetylated and detyrosinated tubulin, whereas reexpression of EB1 in these cells fully rescued the expression of acetylated and detyrosinated tubulins, which accompanied the restoration of vascular lumen formation. (C) Endothelial cells were induced to express GFP, EB1 (wt), EB1 (K220A), or EB1 δ C using recombinant adenoviral vectors and suspended within collagen matrices for 24 hours in 3D collagen matrices. Cultures were fixed with glutaraldehyde, stained with toluidine blue, and photographed. Scale bar is 100 μm. (D) EB1 or control siRNA-treated cells were induced to express control GFP, EB1 (wt), EB1 (K220A), or EB1 δ C, suspended within collagen matrices and allowed to undergo EC tube morphogenesis for 24 hours. Fixed and stained cultures were quantitated for lumen formation. Data are presented as lumen area ± SD (n = 3). Statistical significance *P < .05, **P < .005, ***P < .0005.