Autoinflammatory disease-associated mutations in CIAS1 induce rapid cell death. (A) As the controls for cell death, 1 × 10⁶ THP-1 cells were treated with 1 μg/mL actinomycin D (ActD) and 20 μM nigericin. 0.5 μg of plasmid encoding GFP-CIAS1 or the disease-associated mutants (R260W, D303N, Y570C) were introduced into 1 × 10⁶ THP-1 cells. After transfection (3 hours), cells were analyzed by flow cytometry, and the expression of CIAS1 was monitored by measuring GFP fluorescence. Cell death was assessed as positive annexin V staining (apoptosis) or double positive for annexin V and 7-AAD (necrosis). The number in each quadrant shows the percentage of cells. (B) HEK293 cells were cotransfected with 20 ng of WT-CIAS1 or the indicated mutants in the presence or absence of 20 ng of ASC. The ability to induce NF-κB activation was assessed by a dual luciferase reporter assay in HEK293 cells. Disease-associated mutants induced ASC-dependent NF-κB activation spontaneously. Values represent the mean of normalized data (mock without ASC = 1) of triplicate cultures, and error bars indicate SD. (C) Expression of GFP-CIAS1 and annexin V and 7-AAD staining were monitored by flow cytometry. Expression of GFP was observed at 1 hour and 30 minutes after transfection, at which point annexin V/7-AAD–positive cells started to appear in the Y570C-transfected cell population. The percentage of Y570C-transfected cells that were annexin V/7-AAD positive increased in a time-dependent manner. Representative data from 3 independent analyses of similar results are shown.