ARC is a negative regulator of apoptosis. 293T cells were transfected with pcDNA3, pcDNA3-ARC-Flag, and various expression plasmids as described in Material and Methods. Transfected cells were visualized with β-galactosidase substrate and scored for morphological feature of apoptosis. (A) Caspases were cotransfected with ARC (▪) or without ARC (□). In the experiment, 0.2 μg of plasmid DNA expressing caspase-4, -8, or -10 or 0.1 μg of Ced-3 or caspase-9 plasmid was used in the presence of 2 μg of pcDNA3-ARC-Flag or pcDNA3. (B) ARC inhibits caspase-induced apoptosis in a dose-dependent manner. 0.2 μg of plasmid expressing caspase-4, -8, -10, or pcDNA3 was used. The x axis represents amount of ARC plasmid DNA. Total amounts of plasmid DNA was 2.2 μg in all experiments presented in A and B. (C) ARC inhibits FADD, TRADD, and CLARP-induced apoptosis. The amount of plasmid DNA were: FADD (0.4 μg), TRADD (0.1 μg), CLARP (2.0 μg), or pcDNA (2.0 μg) in the presence of 2.0 μg of ARC or pcDNA3 plasmid. (D) ARC inhibits apoptosis induced by death receptors. The amount of plasmid DNA were: Fas (1.5 μg), TNFR1 (0.2 μg), TRAMP (1.0 μg), or pcDNA3 (2.0 μg) in the presence of 2.0 μg of ARC or pcDNA3 plasmid. Total amounts of plasmid DNA was 4.0 μg in all experiments presented in C and D.