The role of ASC and NALP3 in the production and release of IL-1β. THP-1 cells were transfected with siRNA against ASC or NALP3 or control scramble (siC). RT-PCR of lysates after overnight incubation for ASC (A) or NALP3 (B). Cells were transfected with scrambled or target siRNA, then primed for 24 hours with PMA (100 ng/mL), and thereafter stimulated for 24 hours with LPS (1 μg/mL). After 24 hours, IL-1β (C) or IL-8 (D) was assessed in the supernatants (*P < .05 compared with the stimulation with LPS in cells treated with scramble siRNA). (E) Levels of supernatant IL-1β and TNF-α in primary PBMCs after transfection with scramble or siRNA for ASC and subsequent stimulation with LPS for 24 hours (n = 5, mean ± SEM, *P < .05 compared with LPS/scramble). (F) Levels of supernatant IL-1β in PBMCs stimulated for 3 hours with LPS (100 ng/mL), after which ATP (1 mM) or a combination of LPS and ATP was added for an additional 15 minutes. PBMCs were stimulated that were transfected with either scramble or siRNA against ASC (n = 5, means ± SEM, *P < .05 compared with LPS/ATP in the cells transfected with scramble siRNA). (G) Western blot of ASC in PBMCs transfected with either scramble or siRNA against ASC. (H) Western blot of the p10 and p45 caspase-1 in PBMCs transfected with either scramble (column 1) or siRNA (column 2) against ASC.