Ac yvad cmk

To induce differentiation of THP-1 cells (as well as variants) into macrophages (dTHP-1 cells), the cells were pretreated with phorbol 12-myristate 13-acetate (PMA, P1585, Sigma) (50–100 nM) overnight at 37 °C. All stimulation was performed in reduced serum medium Opti-MEM (31985-070, Gibco). The differentiated cells were washed with PBS and incubated with various concentrations of CytK for 1 h. To examine the effects of various inhibitors on CytK activity, the following inhibitors were used to treat dTHP-1 prior to CytK stimulation: Ac-DEVD-CHO (A0835, Sigma), Ac-YVAD-cmk (SML0429, Sigma), Z-VAD-FMK (S7023, Selleck), necrosulfonamide (NSA, S8251, Selleck), necrostatin-1 stable (Nec-1s, S8641, Selleck), BAPTA-AM (S7534, Selleck), R59022 (B6967, ApexBio), or potassium chloride (KCl, 10016318, Sinopharm). To examine the effect of nigericin (serving as a positive control), the cells were treated as above, except that CytK was replaced by 20 μM nigericin (Nig, 481990, Sigma). For the antibody neutralization assay, CytK was pre-incubated with the antibody against CytK (anti-CytK IgG) or the control antibody (rabbit IgG) (D110502, Sangon Biotech) for 1 h before incubation with dTHP-1 cells.

LPS (Sigma) was dissolved in sterile deionized water and used at a final concentration of 0.1 μg/ml or 1 μg/ml. The caspase-1 inhibitor Ac-YVAD-CMK (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 50 μM. BAY11-7082 (Selleck, USA), as an inflammasome inhibitor, was dissolved in DMSO and used at a concentration of 5 μM, and the ROS inhibitor N-acetylcysteine (NAC, Sigma, USA) was used at a concentration of 10 mM which dissolved in sterile deionized water.

α,α′-Trehalose 6,6′-glycolipids, AF-2 and TDB, were made up to 1 mM in CHCl₃/MeOH (2/1, v/v), then diluted to 400 μM or 50 μM in isopropanol to give 8 or 1 nmol of glycolipid per 20 μL, respectively. The solutions were then added to 96-well plates (20 µL/well) and the solvent was evaporated within a sterile hood for 18 h.
Bone marrow cells were collected from the tibia and femur of C57BL/6 or Mincle^−/− mice and cultured (250,000 cells/mL) in complete Roswell Park Memorial Institute medium [cRPMI-1640 (Gibco) with 10% heat inactivated fetal bovine serum (Gibco), 100 unit/mL penicillin-streptomycin (Gibco) and 2 mM Glutamax (Gibco)]. Macrophage differentiation was induced by 50 ng/mL GM-CSF (PeproTech) added to the cRPMI. Cells were incubated at 37 °C (5% CO₂) for 8 days (cells fed on days 3 and 6). On day 8, the media was removed, the cells were lifted using Accutase (StemCell), counted and re-seeded in 96-well plates coated with ligands. Where indicated Ac-YVAD-cmk (40 μM, Sigma) or KCl (50 mM) [12 (link)] were added to the cells an hour before, or CY-09 (20 or 40 µM) was added 30 min before adding the cells to ligand-coated plate according to the manufacturer’s protocol. 100 ng/mL LPS (Sigma) or nigericin (10 μM, ChemImpex) were used as controls [29 (link)].

Lipopolysaccharide (LPS; Escherichia coli 0111:B4), ATP, and oxidized ATP (oxATP) were obtained from Sigma (St. Louis, MO, USA), and nigericin was purchased from InvivoGen (San Diego, CA, USA). Ac-YVAD-cmk and MCC950(CP-456773 sodium salt) were purchased from Sigma (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Japan). Calcein AM iwas purchased from Invitrogen (Carlsbad, CA, USA) and 1 mg/ml Propidium iodide solution was purchased from Sigma (St. Louis, MO, USA). 1% Crystal violet solution was purchased from Sigma (St. Louis, MO, USA). NLRP3 antibodies and secondary anti-sheep antibody conjugated to horseradish peroxidase (HRP) were purchased from R&D Systems (Minneapolis, MN, USA). Toll-like receptor 4 (TLR4), P2X7, ASC, IL-1β, and caspase-1 donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Human umbilical vein endothelial cells (HUVECtert) were cultured in Nunclon^TM Delta surface flasks (Bartelt; Graz, Austria) in medium 199 (Gibco; Carlsbad, CA, USA) supplemented with 20% fetal calf serum (FCS) (Sigma-Aldrich; St. Louis, MO, USA), penicillin-streptomycin-amphotericin B (Lonza; Basel, Switzerland), and ECGS-heparin (PromoCell; Heidelberg, Germany) in the 95% humidified atmosphere with 5% CO₂ at 37 °C. Stimulation of cells was performed in endothelial basal medium-2 (EBM-2) (Lonza) supplemented with 2% FCS (Sigma-Aldrich), and 20 mM HEPES (Lonza). The cells were preincubated with an inhibitor solution for 20 min. Then, an agonist solution was added to the cells. After 7 h of the incubation at 37 °C, cell culture supernatants were collected for ELISA. At the end of the experiment, viability of the remaining cells was measured using a tetrazolium salt (XTT) assay. Following inhibitors and agonists used were purchased from Sigma-Aldrich: AZ11645373, A740003, Ac-YVAD-cmk, LPS 005:B5, ATP, BzATP. Recombinant human TNFα was from PeproTech (Rocky Hill, NJ, USA).