R837, activator of cryopyrin-associated inflammasome, induces rapid cell death. (A) Addition of 10 μg/mL imiquimod R837 to WT-CIAS1–transfected THP-1 cells induced rapid cell death, as assessed by annexin V– and 7-AAD–positive staining. This effect was effectively suppressed by CA-074-Me (CA074) treatment. (B) ASC-dependent-activation of NF-κB induced by R837 was assessed using a gene reporter assay in HEK293 cells in the presence or absence of 10 μg/mL R837. NF-κB activity in cells expressing each of the disease-related CIAS1 mutants increased approximately 2-fold after R837 treatment. Values represent the mean of normalized data (mock without R837 = 1) of triplicate cultures, and error bars indicate SD. (C) Addition of 2 μg/mL of MDP to WT-CIAS1– and Y570C-transfected THP-1 cells had only a marginal effect on cell death, as assessed by the percentage of cells that were positive for annexin V and 7-AAD. (D) ASC-dependent activation of NF-κB induced by MDP was assessed using a gene reporter assay in HEK293 cells in the presence or absence of 2 μg/mL MDP. Although MDP induced NF-κB activation, it did not show ASC dependency. Values represent the means of the normalized data (mock without MDP = 1) of triplicate cultures, and error bars indicate SD. Representative data from 3 independent analyses of similar results are shown.