TNFR1 deficiency rescues cell death and vessel regression in TAK1^ecko. (A) TUNEL (green) and PECAM-1 (red) staining of control, Tak1^flox/floxTnfr1^−/−, TAK1^ecko TNFR1 heterozygous knockout, Tie2-Cre Tak1^flox/floxTnfr1^+/− and double knockout, Tie2-Cre Tie2-Cre Tak1^flox/flox Tnfr1^−/−yolk sacs at E10.5. Arrows indicate TUNEL positive cells. Scale bar indicates 40 μm. The percentages of TUNEL⁺ cells in the total cell population of yolk sacs are shown. Data are shown as means ± SD (n = 3). ***P < .001. (B) Vessel regression was determined by collagen IV and PECAM-1 double staining at E10.5. The number of vessel regions that were positive for collagen IV but negative for PECAM-1 were defined as regressing vessels and were counted in 30 randomly chosen areas (total in 2 mm²) from 3 different yolk sacs (graph). Data are shown as means ± SD (n = 3). N.S. indicates not significant. (C) Whole-mount PECAM-1 staining of control TNFR1^−/− (Tak1^flox/floxTnfr1^−/−) and TAK1^ecko TNFR1^−/− (Tie2-Cre Tak1^flox/floxTnfr1^−/−) yolk sacs at E10.5. Vessel lengths and branch points were determined in 3 randomly chosen areas from 3 different yolk sacs (graphs). Data are shown as means ± SD (n = 3). N.S. indicates not significant. (D) HUVECs were treated with control siRNA or siRNA targeting Tak1. The protein level of TAK1 is shown in supplemental Figure 9B. Cells were preincubated with vehicle (DMSO) or necrostatin-1 (20μM) for 1 hour and stimulated with 200 ng/mL of TNF for 6 hours. Cells were subjected to 7-amino-actinomycin D (7-AAD) analysis by FACS.