Jam-B and Jam-C are required for Cdc42-dependent signal transduction events and vascular guidance tunnel formation during EC tubulogenesis in 3D collagen matrices. (A) ECs were treated with the indicated siRNAs and were then seeded within 3D collagen matrices to undergo tube morphogenesis. After 16 hours of culture, EC lysates were prepared and analyzed for the indicated molecules. In each case, we have analyzed for phosphorylated kinase as an indicator of kinase activation as well as the total levels for each kinase. (B) EC lysates from the different siRNA conditions were prepared at the indicated time points and were blotted for phospho-Erk1/2 as well as total Erk at the 24-hour point. (C) ECs were treated with the indicated siRNAs and were cultured in 3D collagen matrices for 24 hours. Cultures were fixed with paraformaldehyde and immunostained with an antibody to rat collagen-type I that selectively recognizes native collagen. Arrows indicate vascular guidance tunnels that represent physical spaces created by proteolysis during the EC lumen and tube formation process. Bar equals 50 μm. (D) Quantitation of vascular guidance tunnel formation from cultures described in panel C. Cultures were fixed at 16 and 24 hours, and tunnels were quantitated by tracing areas using Metamorph software. Data are shown as the average tunnel area per HPF ± SD. n = 6, P < .01. * indicates a significant decrease. (E) ECs were treated with the indicated siRNAs and cultures were established in 3D collagen matrices. After 16 hours, detergent lysates were prepared and incubated with GST-PAK-PBD beads as described.16 Eluates were analyzed for Cdc42 activation since these beads only bind Cdc42-GTP. Western blots were probed with antibodies to the indicated components of the lumen signaling complex. Starting material was probed for total Cdc42, which was equivalent in all cases.
