Cathepsin b inhibitor ca074

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.

The renal fibroblasts and the renal epithelial cell lines HK-2 and A498 cells were stimulated with CFT073 wild-type and mutants at a multiplicity of infection (MOI) of 1 or 10 for 6 h and incubated at 37 °C with 5% CO₂. The fibroblasts were also pre-incubated with DMSO (vehicle), 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, Farmingdale, NY, USA) and the PKC inhibitor bisindolylmaleimide I (10 µM, Santa Cruz Biotechnology Inc.), JNK inhibitor SP600125 (10 μM, InSolutionT M JNK Inhibitor II, Calbiochem, San Diego, CA, USA), serine protease inhibitor 3,4-Dichloroisocoumarin (DCI, 100 μM, Merck Millipore, MA, USA), cathepsin B inhibitor CA074 (100 μM, Apexbio Technology LLC, Houston, TX, USA), NLRP3 inhibitor MCC950 (2 μM, Avistron Chemistry Services, Cornwall, UK), Disulfiram (10 μM, Selleckchem, Houston, TX, USA), caspase-3 inhibitor AC-DEVD-CHO (10 μM, Enzo Life Sciences) for 1 h prior to CFT073 stimulation for 4 or 6 h at MOI 1. Supernatants and total RNA were collected and kept at −80 °C until further analysis.