TAK1 signaling is important for tube formation and endothelial cell migration. (A) HUVECs were treated with control siRNA or siRNA targeting Tak1. Tube formation was observed with increasing concentrations of serum after 10 hours. Scale bar indicates 50 μm. Cells were visualized by Calcein AM, and total lengths of all formed tubes and branch points were measured. The lengths and branch points in TAK1-knockdown cells relative to those in controls are shown. Data are shown as means ± SD (n = 3). ***P < .001; **P < .01. The levels of TAK1 protein were analyzed by immunoblot. (B) HUVECs were treated with control siRNA or siRNA targeting Tab2. Tube formation was observed in response to serum after 10 hours. Scale bar indicates 50 μm. Total lengths of all formed tubes and branch points were measured, and the lengths and branch points in TAB2-knockdown cells relative to those in controls are shown. Data are shown as means ± SD (n = 5). ***P < .001 The levels of TAB2 protein were analyzed by immunoblotting. (C) The migration of TAK1- and TAB2-knockdown HUVECs was assayed using Transwells coated with collagen type 1. HUVECs were suspended in the medium and seeded in the upper chamber and the medium with or without 8% serum was added to the lower chamber. Cells were allowed to migrate across an 8.0-μm pore size membrane for 6 hours and stained with DAPI and the percentages of migrating cells in the total cell population were determined. Data are shown as means ± SD (n = 4). ***P < .001; **P < .01.