Signaling events that characterize EC tubulogenesis over time in 3D collagen matrices.

<p>(A) ECs suspended in 3D collagen matrices using the Factor-induced model were lysed at the indicated time points for Western Blot analysis to assess expression and signaling of the indicated molecules or were fixed and photographed while ECs are assembling into tubes over time. Bar equals 50 μm. (B) The same time courses were analyzed for phosphotyrosine (pTyr)-containing proteins and increased levels of pTyr bands at 225, 150, 130, 120, 100, 76, 68, 60, 45, 40, 36, and 30 kDa were identified over time correlating with the EC tubulogenic process. (C) ECs were infected with the indicated recombinant adenoviruses and then a lumen assay was performed over 72 hr. Upper panel- ECs were infected with mCherry or PKCɛ adenoviruses, then cultured in 3D collagen gels using the Factor-induced model, fixed and photographed. Bar equals 50 μm. Lower panel- ECs were infected with adenoviruses carrying GFP, Csk, DN Csk, PKCɛ, tail-deleted catalytically active WT MT1-MMP (MT1-ΔCT wt) or a dominant negative MT1-MMP construct (MT1-ΔCT EA), then suspended in 3D collagen matrices to assess their ability to form lumens. Data are reported as the mean vessel area ± SD per HPF (n = 12; p < 0.01). Asterisks indicate significance compared to control GFP cultures.