P0883

Primary murine BMDMs were seeded in 96-well plates overnight before use (1x10⁶ cells/ml). Cells were primed with lipopolysaccharide (LPS; E.coli O26:B6) (1 μg mL⁻¹) (Sigma, L2654) in DMEM (10% v/v FBS, 1% v/v penicillin–streptomycin, 1% v/v pyruvate) for 4 h, before the media was replaced with ‘treatment media’ constituting DMSO (1% v/v) (Sigma, D2650) or brazilin (≥98% purity; HPLC) (Merck, SML2132; Rahway, NJ, USA) (30, 10, 3, 1, 0.3, 0.1, 0.03 or 0.01 μM) in serum-free DMEM (1% v/v penicillin–streptomycin, 1% v/v pyruvate). After 15 min treatment incubation, NLRP3 was activated by adding nigericin (10 μM) (Sigma, N7143) into the wells, with ethanol (0.5% v/v) used as a vehicle control, and incubated for 2 h. Alternatively, to assess the direct effect of brazilin treatment on AIM2 or NLRC4 inflammasome activation, cells were transfected with either 1 μg mL⁻¹ poly(deoxyadenylic-deoxythymidylic) acid sodium salt (poly(dA:dT), Sigma, P0883) or 1 μg mL⁻¹ flagellin from Salmonella typhimurium (Invivogen, tlrl-stfla), respectively, using Lipofectamine 3000 (Thermofisher, L3000001) diluted in OptiMEM (Thermofisher, 11058021) per manufacturer’s instructions, for 2 h. Following 2-h inflammasome activation, supernatants were collected and used for cell death analysis and Enzyme-Linked Immunosorbent Assays (ELISA).