Mcc950

IL-1β and TNF-α ELISA kits were purchased from eBioscience (San Diego, CA, USA) or R&D Systems (Minneapolis, MN, USA). Silica crystals (nano-SiO₂), nigericin, poly(dA:dT), flagellin, and carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-FMK) were purchased from InvivoGen (San Diego, CA, USA). LPS (0111:B4), ATP, L-cysteine, glutathione, and forskolin were obtained from Sigma-Aldrich (St. Louis, MO, USA). H89 was purchased from Alexis Biochemicals (San Diego, CA, USA). Lipofectamine 2000 and disuccinimidyl suberate (DSS) were acquired from Thermo Fisher Scientific (Rockford, IL, USA). G5 was purchased from EMD Millipore (Bedford, MA, USA). The lactic dehydrogenase (LDH) detection kit was from Dogen EINNOTEC (Dae Jeon, Korea). MCC950 was purchased from AdipoGen (San Diego, CA, USA). The following antibodies were used for western blotting: anti IL-1β, (AF-401-NA, R&D Systems, Minneapolis, MN, USA); NLRP3 (Cryo-2), ASC (AL-177), and caspase-1 (AG-20B-0042-C100) from AdipoGen (San Diego, CA, USA); ASC (N-15), caspase-1 (SC-514), and β-actin from Santa Cruz Biotechnology (Dallas, TX, USA).

MCC950 (Adipogen, San Diego, CA), glycine (Fisher Scientific, Waltham, MA) and potassium chloride (Fisher Scientific) were resuspended in deionized water. Go6983 (Selleck Chemicals, Houston, TX), Ac-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK or YVAD) (Cayman Chemical, Ann Arbor, MI), MI2 (Tocris Bioscience, Bristol, UK), PS1145 (Cayman Chemical), R406, and entospletinib (Selleck Chemicals), were all resuspended in DMSO. Monocytes were treated with the inhibitors or with an equivalent volume of the appropriate vehicle, for 40 min at 37°C and then infected or stimulated as described above. MCC950, Go6983, MI2, PS1145, R406 and entospletinib were all added to infected cells in half log titrations to determine the concentrations of the inhibitors that did not induce cell death or reduce infection efficiency.

Mice were euthanized by CO₂ asphyxiation.The colons were dissected and removed from C57BL/6 and Winnie mice (n = 5, 12 weeks). The colon was opened and the faecal matter removed and cut in half lengthways, sectioned into distal and proximal tissue and weighed. Tissues are washed in PBS (P3813 Sigma) containing 1% penicillin/streptomycin (10000 U/ml) (1% P/S) (Gibco 15140122) three times. Equivalent amount of tissue were placed in a 24 well cell culture plate in growth media RPMI 1640 supplemented with 10% FCS, 1% P/S. The tissues were stimulated with 10 ng/ml Lipopolysaccharide (LPS) from Escherichia coli serotype EH100 (ra) TLRgrad for 2 hours. The medium was removed and replaced with serum-free medium (SFM) containing MCC950 (0.001–10 μM) (Adipogen), glyburide (200 μM) (Sigma-Aldrich) and incubated for 24 hours at 37 °C in a moist atmosphere of 5% CO₂. After which the supernatants were removed and centrifuged at 12,000 g at 4 °C for 15 min and stored at −80 °C for cytokine analysis. Tissue was stored in RIPA buffer with protease inhibitor to be analysed by western blot. Supernatants were assayed for cytokine levels by ELISA kits according to the manufacturer’s instructions IL-1β (DuoSet, R&D Systems) IL10 (BMS614-2 Invitrogen), TNF-α (KMC3011 Invitrogen) and concentrations were normalized to the weight of the explants.

Primary monocytes were seeded into 96‐well plates at 5 × 10³ cells/well, in RPMI 1640, GlutaMAX medium (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (Lonza). When indicated, primary monocytes were incubated for 3 h in the presence of LPS (10 ng/ml, InvivoGen). Unless otherwise indicated, primary monocytes were then treated for 90 min with nigericin (5 μM, InvivoGen), ATP (2.5 mM, Sigma) (Mariathasan et al, 2006), staurosporine (1.25 μM, Tocris), UCN‐01 (12.5 μM, Sigma), or Ro31‐8820 (100 μM, Tocris). When indicated, monocytes were treated with colchicine (1 μM or at the indicated concentration, Sigma), MCC950 (10 μM, Adipogen AG‐CR1‐3615), paclitaxel (Taxol, 5 μM, Sigma), nocodazole (5 μM, Sigma), z‐YVAD‐FMK, z‐IETD‐FMK, z‐DEVD‐FMK (at the indicated concentrations, Bachem, #4027532, #4034771, #4027402, respectively), VX‐765 (InvivoGen), 30 min before addition of UCN‐01, TcdB (Abcam, #ab124001, 125 ng/ml), and TcdA (1 μg/ml) or nigericin. TcdA was purified from Clostridioides difficile VPI10463 strain, as previously described (von Eichel‐Streiber et al, 1987; Popoff, 1987). Following the incubation, cells were centrifuged and supernatants were collected.

The CASP1 activity for the determination of inflammasome activation was determined by using the Caspase-Glo^® 1 Inflammasome Assay (Promega, Madison, WI, USA), following the manufacture’s protocol. In brief, half of the supernatant was removed and replaced with the 1:2 dilution of the ready-to-use Glo1 reagent and incubated for 60 min at room temperature. For NLRP3 inhibition, the cells were pre-incubated for 1 h with the NLRP3-specific inhibitor MCC950 (1 µM; AdipoGen Life Science, Liestal, Switzerland). Finally, luminescence signals were read out with the Tecan Infinite^® M200 plate reader.

The transduced THP-1 cells were differentiated with 200 nM PMA for 24 hr and treated with DOX (Wako) or nigericin (InvivoGen) at the indicated concentrations. Next, cells were cultured at 37 or 32°C. Cells were then pretreated with inhibitors including CA-074 (Wako), MCC950 (Adipogen, San Diego, CA), probenecid (Cayman, Ann arbor, MI), trovafloxacin mesylate (Cayman), VX-765 (Selleck), and Z-VAD-FMK (MBL) for 30 min prior to cold exposure.