All compounds were dissolved in DMSO (D2650, MilliporeSigma) and diluted with cell culture media. For apoptosis inhibitors, a pan‐caspase inhibitor z‐VAD‐FMK (FMK001, R&D systems) was used at 50 μM, z‐DEVD (FMK004, R&D systems), targeting Caspase‐3, ‐6, ‐7, ‐8, and ‐10, was used at 25 μM, Wedelolactone (401474, Calbiochem), targeting Caspase‐11, was used at 10 μM, z‐WEHD‐FMK (FMK002, R&D systems), targeting Caspase‐1, ‐8, and ‐11, was used at 20 μM, VX‐765 (S2228, Selleckchem), targeting Caspase‐1 and ‐4, was used at 20 μM, z‐YVAD‐FMK (218746, MilliporeSigma), targeting Caspase‐1 and ‐11, was used at 20 μM, z‐VEID‐FMK (FMK006, R&D systems), targeting Caspase‐6, was used at 10 μM. For necroptosis inhibitors, Necrostatin‐1 (N9037, MilliporeSigma), targeting RIPK1, was used at 50 μM, and GSK872' (6492, R&D systems), targeting RIPK3, was used at 2 μM. Both inhibitors and vehicle (DMSO) were added to the cell culture medium 1–2 h prior to the treatment of MCM and control media.
Gsk 872
Manufactured by R&D Systems
Sourced in United States
GSK'872 is a small molecule inhibitor developed for research purposes. It functions as a selective inhibitor of the LRRK2 (leucine-rich repeat kinase 2) enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Z veid fmk, by R&D Systems (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Vx 765, by Selleck Chemicals (1 mentions)
Z wehd fmk fmk002, by R&D Systems (1 mentions)
D2650, by Merck Group (1 mentions)
Necrostatin 1, by Merck Group (1 mentions)
Sb203580, by Santa Cruz Biotechnology (1 mentions)
Pd98059, by Santa Cruz Biotechnology (1 mentions)
Ac yvad cho, by Enzo Life Sciences (1 mentions)
Nf κb inhibitor bay 11 7082, by Enzo Life Sciences (1 mentions)
3 4 dichloroisocoumarin dci, by Merck Group (1 mentions)
Ac devd cho, by Enzo Life Sciences (1 mentions)
Cathepsin b inhibitor ca074, by Apexbio (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Necrosulfonamide nsa, by Bio-Techne (1 mentions)
Nec 1, by Merck Group (1 mentions)
Primepcr sybr green assay, by Bio-Rad (1 mentions)
Mki67, by Bio-Rad (1 mentions)
4 protocols using gsk 872
1
Apoptosis and Necroptosis Inhibitor Assay
2
Neutrophil Response to Uropathogenic E. coli
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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 1015 (link). mRNA, proteins and supernatants were collected and kept at − 80 °C until further analysis.
3
Cigarette Smoke Extract Cytotoxicity Assay
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CSE was prepared using Kentucky 2R4F research-reference filtered cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, Kentucky, USA) as previously described (43 (link)). One cigarette was used to prepare 10 mL of the cell growth medium, which was regarded as 100% CSE. The epithelial cells were seeded in 12-well plates or 96-well plates 24 hours before CSE treatment. The organoids were subcultured in a 48-well plate for 6 days prior to CSE treatment.
The chemicals used include Vp-16, 3 MA, cycloheximide (MilliporeSigma), Z-VAD (Bachem), TNF-α, Nec-1, and GSK’872 (R&D Systems, Bio-Techne). TNF-α was dissolved in H2O. The other agents were dissolved in DMSO (MilliporeSigma) for stock.
The chemicals used include Vp-16, 3 MA, cycloheximide (MilliporeSigma), Z-VAD (Bachem), TNF-α, Nec-1, and GSK’872 (R&D Systems, Bio-Techne). TNF-α was dissolved in H2O. The other agents were dissolved in DMSO (MilliporeSigma) for stock.
4
Extraction and Analysis of CV Bioactive Compounds
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The CV extract was prepared from commercially available CV capsules (MycoMedica Company, Police nad Metují, Czech Republic) following previously described protocols [38 (link),40 (link),41 (link)]. The protein and carbohydrate contents in this extract were measured using phenol sulfuric acid and bicinchoninic acid assays, respectively. d -pen, KA (5-hydroxy-2-hydroxymethyl-1,4-pyrone), melanin, Nec-1, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and lipopolysaccharide (LPS) were obtained from Sigma-Aldrich (Darmstadt, Germany). GSK’872 was purchased from R&D Systems (Minneapolis, MN, USA), and necrosulfonamide (NSA) was purchased from Tocris Bioscience (Bristol, UK). An EXTRACTME Total RNA Plus kit was obtained from Blirt company (Gdansk, Poland), and iScript Reverse Transcription Supermix for RT-qPCR, SSoAdvanced Universal SYBR® Green Supermix, the PrimePCR™ SYBR® Green Assay, human IL-1β (qHsaCID0022272), IL-2 (qHsaCID0015409), IL-6 (qHsaCID0020314), IL-12 (qHsaCID0007212), MKI67 (qHsaCID0011882), ITGA5 (qHsaCED0045939), ITGB3 (qHsaCED0045078) and ACTB (qHsaCED0036269) were purchased from Bio-Rad (Hercules, CA, USA). Ficoll–Paque Plus was bought from Amersham Biosciences (Piscataway, NJ, USA). All reagents for cell culture were purchased from Sigma-Aldrich unless stated otherwise.
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