Suppression of NLRP3-speck formation by CARD8. (A) Human Embryonic Kidney (HEK)293 cells were cotransfected with expression plasmid constructs of NLRP3-GFP, caspase recruitment domain-containing protein 8 (CARD8)-DsRed, procaspase-1, and either apoptosis-associated speck-like protein containing a CARD (ASC) or an empty vector. After 24 hours, green and red fluorescent signals were detected by a confocal laser scanning microscope. The scale bar represents 10 μm. (B) The same as A except for using NLRP3, CARD8-GFP and procaspase-1-DsRed. (C) The same as A, except for using NLRP3-GFP, CARD8 and procaspase-1-DsRed. (D) HEK293 cells were cotransfected with expression plasmids for NLRP3-GFP (30 ng), ASC (5 ng), procaspase-1 (2.5 ng), proIL-1β (30 ng) and CARD8 (5, 15, 50, 150 or 500 ng) or an empty vector. At 24 hours after transfection, the percentage of NLRP3-GFP-speck positive cells in the transfected cells was determined. Data represent the mean ± SD (n = 9).