Ac yvad cho

At 3 h prior to infection, the medium was changed to complete medium (RPMI) plus 1% fetal bovine serum with or without 100 ng/ml ultrapure lipopolysaccharide (LPS; Sigma, Shanghai, China). The PMs were stimulated with N. caninum for the indicated times (1–24 h) at various multiplicities of infection (MOI = 1:1, 3:1, and 5:1). PMs treated with medium alone or LPS were used as negative controls, and cells treated with ATP (5 mM, 30 min; Sigma, Shanghai, China) were used as positive controls, as extracellular ATP is a conventional NLRP3 inflammasome agonist [14 (link)].
To monitor the role of the inflammasome in response to N. caninum infection in PMs, PMs were pre-treated with 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Lausen, Switzerland), 100 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai, China) or 100 μM glyburide (an inhibitor of NLRP3 by inhibiting K⁺ efflux; Selleck, Shanghai, China) for 45 min before stimulation, and the PMs were then stimulated with LPS priming plus N. caninum for 12 h at an MOI of 3:1 (parasite:cell), and PMs cultured with 0.2% DMSO were used as a negative control.

To monitor the roles of activated TLR and NLR in response to GEVs and G.duodenalis infection in murine peritoneal macrophages, cells were pretreated with 50 μM C29 (an inhibitor of TLR2 by blocking hTLR2/1 and hTLR 2/6 signal, MedChemExpress, USA), 50 μM Glibenclimide (an inhibitor of NLRP3 by inhibiting K⁺ efflux; Selleck, Shanghai), or 25 μM CA-074 methyl ester (CA-074 Me, an inhibitor of cathepsin B, MedChemExpress, USA) for 1 h before stimulation. The mRNA fold change of activated TLR and NLR and downstream inflammatory cytokines were determined using qPCR, and inflammatory cytokines protein levels were determined using ELISA and western blot.
To monitor the role of activated inflammasome in response to GEVs and G. duodenalis infection in murine peritoneal macrophages, cells were pre-treated with 50 μM Glibenclimide, 25 μM CA-074 methyl ester, 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Switzerland), or 10 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai) for 1 h before stimulation. The mRNA fold change of activated NLR/caspase-1 and downstream inflammatory molecules were determined using qPCR, and inflammatory molecules protein levels were determined using ELISA and western blot.

Human neutrophils were stimulated with the wild type CFT073 or CFT073 mutant bacteria for 3 or 6 h at a multiplicity of infection (MOI) of 1 or 10 and incubated at 37 °C with 5% CO₂. Neutrophils were also pre-incubated with caspase-1/4 inhibitor AC-YVAD-CHO (10 µM, Enzo Life Sciences, NY, USA), caspase-3 inhibitor AC-DEVD-CHO (10 µM, Enzo Life Sciences), NLRP3 inhibitor MCC950 (2 µM, Avistron Chemistry Services, Cornwall, UK), JNK inhibitor SP600125 (10 µM, InSolutionT M JNK Inhibitor II, Calbiochem, USA), p38 MAPK inhibitor SB203580 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), ERK1/2 inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc), NF-κB inhibitor BAY 11–7082 (5 µM, Enzo Life Sciences), serine protease inhibitor 3,4-Dichloroisocoumarin (DCI, 100 µM, Merck Millipore, MA, USA), cathepsin B inhibitor CA074 (100 µM, Apexbio Technology LLC, Houston, USA), actin polymerization inhibitor cytochalasin D (Cyto D, 10 µg/ml, Santa Cruz Biotechnology Inc), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) inhibitor GSK-872 (10 µM, R&D Systems, Minneapolis, USA) or DMSO as vehicle control, for 1 h prior to CFT073 stimulation for 3 or 6 h at MOI 1 or MOI 10^{15 (link)}. mRNA, proteins and supernatants were collected and kept at − 80 °C until further analysis.

PMφ was pretreated with either 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Switzerland) or 10 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai) for 1 h before A. sobria stimulation (MOI=10) to explore the roles of caspase-1 p20 in activated IL-1β secretion (Malyshev et al., 2004 (link); Cao et al., 2005 (link)). PMφs were pretreated with either 25 μM CA-074 methyl ester (CA-074 Me, an inhibitor of cathepsin B, MedChemExpress, USA) or 50 μM Glyburide (an inhibitor of NLRP3 by inhibiting K⁺ efflux; Selleck, Shanghai) for 1 h before stimulation to block NLRP3 receptor activation and explore its roles in caspase-1 p20 production and IL-1β p17 secretion (Lamkanfi and Dixit, 2009 (link); Liu et al., 2013 (link)). PMφs were pretreated with these NLRP3 inflammasome inhibitors through different ways to detect its roles in inflammatory response by measuring cytokines levels of IL-1β, IL-6, IL-12, and TNF-α.