Mutant TNFR1 do not induce increased spontaneous signaling compared with wild-type TNFR1. (A-B) HT1080 cells were transfected with the indicated full-length TNFR1 or TNFR1-ΔCD or YFP expression vectors and allowed to incubate for 18 hours. Some cells were incubated with 50 μM zVAD-fmk to inhibit caspase activation or 0.1 μM staurosporine (STS) as a positive control for cell death. Cells were then stained with TMRM and analyzed by FACS. (A) Insets indicate percent YFP⁺TMRM⁺ (live) cells over YFP⁺TMRM^– (dead) cells. (B) Percentage of cell death was determined by first gating on the YFP^med population and then the TMRM^– cells (inset). Error bars indicate SD. (C) HT1080 cells were transfected with the indicated full-length TNFR1 or TNFR1-ΔCD expression constructs along with an NF-κB luciferase reporter and the renilla luciferase (RLTK) as a transfection efficiency control. Cells were also incubated with 50 μM zVAD-fmk to prevent spontaneous cell death. Twenty-four hours later, luciferase expression was examined and NF-κB induction was normalized by comparing it with the renilla luciferase intensity of each sample (±SD). Experiments shown were performed in duplicate, and data are representative of multiple experiments. *P < .05 between WT and mutant TNFR1 constructs by an unpaired Student t test.