N7143

The quinolinium salt-based halide-sensitive fluorescence probe N-(ethoxycarbonylmethyl)-6-methoxyquinolium bromide (MQAE; ab145418, Abcam) was used as a measure of chloride ion influx activity13 (link). Following cell culture, the PRNCs were incubated with 5 mM MQAE for 2 h at RT in the dark, and subsequently washed twice with NbActive 4 (BrainBit). Cells were then treated with 50 μM GABA (A2129, Sigma-Aldrich) or 10 nM GABA analog THIP, δ-GABA_AR specific agonist8 (link) (T101, Sigma-Aldrich), then fluorescence intensity was consequently measured at 0, 5, 10, and 20 min at RT. For inhibition assay, the cells were pretreated with 1 μM flumazenil (F6300, Sigma-Aldrich) or 1 μM picrotoxin (P1675, Sigma-Aldrich) or PBS (control) for 45 min at RT, and then stimulated with 50 μM GABA for 10 min at RT. Intercellular MQAE is quenched by 10 μM tributyltin chloride (T50202, Sigma-Aldrich) and 10 μM nigericin sodium salt (N7143, Sigma-Aldrich). The fluorescence intensity was measured by the Gemini EX florescence plate reader (Ex/Em = 360/460; Molecular Devices). The kinetic analysis was performed by using GraphPad Prism 6^® software.

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).

Pyroptosis in HUVECs was measured using FAM Caspase-1 Kit based on FLICA technology to detect caspase-1 activation (Bio-Rad Laboratories GmbH; ICT9146). FAM–VAD–FMK was dissolved in dimethyl sulfoxide (DMSO; Sigma) following the manufacturer’s instructions. HUVEC cells were seeded in 6 well cell culture plates (657160; Greiner Bio-One) and stimulated after 24 h with Nigericin (10 µM; N7143; Sigma Aldrich) or IL-1β (100 ng/mL; 201-LB; R&D Systems) for 3 h at 37 °C. HUVEC were detached with Accutase® Cell Detachment Solution (A6964-100ML; Sigma Aldrich), centrifuged (500×g, 5 min), and resuspended in HUVEC medium. FLICA staining was then prepared by adding 5 μl of FLICA working solution into 295 μl of culture medium. Cells were incubated for 30 min at 37 °C. Following two washing steps, HUVECs were resuspended in 500 µl 1× washing buffer and stained with 5 µL 7AAD (420404; Biolegend), prior to acquisition on BD FACS Canto II flow cytometer. The data were analyzed using FlowJo (Version 10; FlowJo LLC).