Cell culture and transfection

HEK293T cells stably expressing FLAG‐tagged pro‐caspase 1 (pro‐caspase 1‐FLAG) and green fluorescent protein (GFP)‐tagged–ASC (designated ASC‐GFP_C1‐FLAG)—as kindly provided by Emad Alnemri (Thomas Jefferson University, Philadelphia, PA)—were cultured in Dulbecco's modified Eagle's medium/Ham's F12 medium (Invitrogen) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml). HEK293T (ASC‐GFP_C1‐FLAG) cells were transfected with either 375, 500, or 750 ng of pNLRP3‐WT or pNLRP3‐A441V or with the empty vector for 24 hours using FuGENE HD (Promega).

THP‐1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with glutamine, penicillin (100 IU/ml), and streptomycin (100 μg/ml). The cells were primed with 100 ng/ml phorbol myristate acetate (PMA) for 3 hours, then transfected either with 500 or 1000 ng of pNLRP3‐WT or pNLRP3‐A441V or with the empty vector using the FF‐100 program in the 4D‐Nucleofector, Amaxa (Lonza). Transfected THP‐1 cells were then distributed in six‐well plates and treated directly with 100 ng/ml lipopolysaccharide (LPS) at 37°C. Twenty‐four hours following transfection and treatment, supernatants were collected and stored at −80°C for cytokine measurement.