Dantrolene

Cells (5 × 10⁴) treated with or without RYR inhibitor (dantrolene, 100 µM, MedChemExpress, Monmouth Junction, USA) were resuspended in 100 µl of PAG-CM buffer (Piperazine-N,N-bis[2-ethanesulfonic acid]-Albumin-Glucose buffer containing 3 mM CaCl₂ and 1.5 mM MgCl₂, pH 7.4) and stimulated with vehicle (spontaneous release), compound 48/80 (c48/80) (5 µg/ml, Sigma-Aldrich, St. Louis, Missouri) or substance P (SP) (30 μM, MedChemExpress, Monmouth Junction, USA) for 60 min. Cells were centrifuged and supernatants (SNs) were collected and the pelleted MCs were rapidly frozen with 100 μl of H₂O. After thawing, 50 μl of SNs or cell lysates were incubated with the same volume of 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide (Sigma-Aldrich, Munich, Germany) solution at 5 μM in citrate buffer (pH 4.5) for 60 min at 37°C. Sodium carbonate buffer (100 mM; pH 10.7) was added to stop the reaction. Fluorescence intensity was determined at excitation at 355 nm and emission wavelength of 460 nm. % β‐hexosaminidase release = [fluorescence intensity SN/(fluorescence intensity SN + fluorescence intensity lysate)] × 100. The net release was calculated by subtracting spontaneous release.

4-Hydroxytamoxifen was purchased from Aladdin (Shanghai, China). Jasplakinolide, taxol and caffeine were obtained from Abcam (Cambridge, UK). 2-Aminoethyl diphenylborinate, dantrolene, Z-VAD-FMK, SP600125, lipopolysaccharide (LPS) and nigericin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MCC950 were obtained from CSNpharm (Chicago, IL, USA). PEG8000 and PF431396 were obtained from Beyotime Biotechnology (Shanghai, China). Cantharidin was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Rabbit anti-caspase-1, rabbit anti-ASC and rabbit anti-NLRP3 antibodies were obtained from Proteintech (Chicago, IL, USA). Mouse anti-α-tubulin antibody was from Boster (BM1452, Wuhan, China). Mouse anti-β-actin was purchased from Cell Signaling Technology (Danvers, MA, USA). The siRNA targeting ASC and CASPASE-1 was constructed by GenePhrama (Shanghai, China). The Flag-Gsdmd-NT, Flag-Gsdmd-CT plasmid were purchased from Addgene (Watertown, MA, USA).