Fig. 3. siRNA suppression of Cdc42 or Rac1 markedly blocks EC lumen and tube formation as well as EC invasion as Cdc42 and Rac1 are highly activated during EC tube and lumen formation in 3D collagen matrices. (A-C) ECs were treated with the indicated siRNAs and were suspended within collagen matrices (A) or on the surface of collagen matrices containing either 1 µM S1P (B) or 200 ng/ml SDF-1 (C). Quantification occurred at either 48 hours (A,C) or 24 hours (B). Data are shown as the mean number of EC lumens per high-power field (HPF) (A) or invading ECs per HPF (B,C) ±s.d. (n=6). ^*, P<0.01 compared with luciferase control. (D) siRNA-transfected EC lysates were prepared for western blot analysis and probed for Cdc42, Rac1, RhoA or beta-actin (control). (E) ECs were suspended in collagen matrices and extracts were prepared at the indicated time-points. Equal amounts of extracts were incubated with GST-PAK-PBD protein agarose beads or control blank beads. Eluates and starting extracts were analyzed by western blot using antibodies against Cdc42 or Rac1, and levels of activated Cdc42 or Rac1 were compared with their total levels. (F) Relative levels of activated Cdc42 or Rac1 and total Cdc42 or Rac1 were quantified by using Scion imaging software. The data are expressed in arbitrary units (AU)±s.d. (n=3).