Primary human monocytes were primed with 1 μg/mL ultrapure LPS and 1μg/mL Pam3CSK4 for 3 h. THP-1 cells were primed with 1 μg/mL Pam3CSK4 for 4 h. Primed cells (1 × 105) were treated for 1 h with DMSO vehicle, 5 μM LDC7559 (unless indicated otherwise), 10 μM MCC950, or 50 μM VX765, and then for 1 h with 10 μg/mL nigericin, or 25 μg/mL LPS (electroporated using the NEON electroporation system; ThermoFisher Scientific), or they were transfected over 3 h with 1 μg/mL poly(dA-dT) using lipofectamine 2000 (0.5 μL/well).
LDH released into the culture medium was measured 1-3 h post-activation using a CellTox Green Cytotoxicity assay (Promega). IL-1β secretion was measured using a MSD Cytokine Assay, 96-well multi-array Tissue Culture Kit (Meso Scale Discovery). For immunoblotting, monocytes (2 × 106) were treated with 10-50 μM LCD7559 or 50 μM VX765 and electroporated with 25 μg/mL LPS using the NEON electroporation system. Cells were lysed after 1 h using lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM MgCl and 1X Roche cOmplete protease inhibitor cocktail) and protein concentration was determined via BCA protein assay (Peirce). Antibodies for immunoblotting were GSDMD (L60, Cell Signaling Technologies), cleaved GSDMD-Asp275 (E7H9G, Cell Signaling Technologies) and GAPDH (14C10, Cell Signaling Technologies).
LDH released into the culture medium was measured 1-3 h post-activation using a CellTox Green Cytotoxicity assay (Promega). IL-1β secretion was measured using a MSD Cytokine Assay, 96-well multi-array Tissue Culture Kit (Meso Scale Discovery). For immunoblotting, monocytes (2 × 106) were treated with 10-50 μM LCD7559 or 50 μM VX765 and electroporated with 25 μg/mL LPS using the NEON electroporation system. Cells were lysed after 1 h using lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM MgCl and 1X Roche cOmplete protease inhibitor cocktail) and protein concentration was determined via BCA protein assay (Peirce). Antibodies for immunoblotting were GSDMD (L60, Cell Signaling Technologies), cleaved GSDMD-Asp275 (E7H9G, Cell Signaling Technologies) and GAPDH (14C10, Cell Signaling Technologies).