Z vad fmk

BOT-4-one was provided by Sang-Kyu Ye and Byung-Hak Kim (Seoul National University of College of Medicine, South Korea)^{17 (link)}. Penicillin-streptomycin, fetal bovine serum (FBS), opti-MEM, and RPMI 1640 were purchased from Gibco (Grand Island, NY, USA). IL-1β antibody (AF401)⁸ and IL-1β enzyme-linked immunosorbent assay (ELISA) kit were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies against ASC (AL177)⁸ , NLRP3 (Cryo-2)⁸ , and caspase-1 (clone Casper-1)⁸ were purchased from Adipogen (San Diego, CA, USA). β-actin (sc-1616)^{43 (link)} and ubiquitin (sc-8017)^{37 (link)} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monosodium urate (MSU) crystal, Bay11-7082, silica crystal, nigericin, flagellin from S. typhimurium, poly(dAdT), Pam3CSK4, and zVAD-FMK were purchased from InvivoGen (San Diego, CA, USA). Mouse TNF-α ELISA Ready-SET-Go was purchased from eBioscience (San Diego, CA, USA). A western blot chemiluminescence reagent kit was purchased from Pierce Chemical (Rockford, IL, USA). Polyvinylidene fluoride (PVDF) and nitrocellulose membrane were purchased from Millipore Corporation (Bedford, MA, USA). LPS (Escherichia. coli 026:B6 and 011:B4), 3,4-methylenedioxy-β-nitrostyrene, and ATP were purchased from Sigma-Aldrich (St.Louis, MO, USA). All other chemicals used were of the highest quality among those that are commercially available.

Stock solutions were prepared in dimethyl sulfoxide (Applichem): MEK1/2 inhibitor trametinib (1 mM; biomol #1187431), disulfiram (1 mM; Sigma Aldrich #97-77-8), ammonium diethyldithiocarbamate (1 mM; Sigma Aldrich #359548), ammonium tetrathiomolybdate (10 mM; Sigma Aldrich #15060), pan-caspase inhibitor Z-VAD-FMK (20 µM; InvivoGen #tlrl-vad), and JUN N-terminal kinase inhibitor SP600125 (100 mM, InvivoGen #tlrl-sp60). Stock solutions were prepared in aqueous buffer: copper (II) D-gluconate (1 mM, Sigma Aldrich #344419) in PBS and N-acetyl-L-cysteine (10 mM; Sigma Aldrich #A7250) in RPMI medium.

Mice received 5mg/kg zVAD-FMK (InvivoGen) I.P. once per day on days 4 and 6 post-IOE infection or on days 4, 5, and 6 post-infection. Mice received 100 μg Necrostatin-1s (Nec-1s; BioVision) by I.P. injection twice per day 3–6 days post-infection, or twice per day 6–9 days post-infection and once per day 10–13 days post-infection. Mice received 10 mg/kg doxycycline by I.P. injection once or twice per day 6–9 days post-infection, and once per day 10–13 days post-infection.

The SeV M and the VSV wild type and mutant (VSV-M) were kindly provided by Dr. Dominique Garcin (Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland). The SindV was kindly provided by Dr. Marco Vignuzzi (Institut Pasteur, Paris, France). Briefly, BMDMs cells were washed once with 1⨯ PBS and incubate with VSV, VSV-M, EMCV, SeV, SeV-M, and SindV at a MOI of 5 for 2 h at 37 C in 5% CO₂. Then, the medium with a non-adsorbed viruses was removed by washing, the cells were washed with serum-free medium and cultured in DMEM supplemented with 5% FBS, at 37 °C in 5% CO₂. After different time post-infection, conditioned media were collected for virus titration, necrosis, and cytokine quantification and some western blot analyses. Whole cell extracts were used for western blot analyses. As a control of virus replication, viruses were UV inactivated by irradiation for 5 min in a Stratagene 2400 UV cross-linker.
In some experiments, BMDMs were treated with zVAD-fmk (10 μM) (Invivogen), Nigericin (10 μM) (Invivogen), ATP (5 mM) (Invivogen), and glyburide (25 μg/ml) (Invivogen). BMDMs were primed with ultrapure LPS (100 ng/ml) (Invivogen).

Cell death was investigated similarly as described above using a high-content imaging approach. In short, KLN205 cells were exposed to 33% Cu-doped TiO₂ NPs for 24 h at 0, 20, 40, 60, 80 or 100 µg/ml in the absence or presence of Z-VAD-fmk (10 µM, InVivoGen, USA). Following NP exposure, cells were washed and stained with cell death dye as described above and analyzed for cell death.

All chemicals used in this study were purchased from Sigma (St. Louis, MO), unless otherwise noted. Ultrapure LPS, ATP, caspase-1 inhibitor ZVAD-FMK were purchased from Invivogen (San Diego, CA). Mouse IL-1β from PeproTech (Rocky Hill, NJ); human or Mouse IL-1β ELISA kits were from R&D system (Minneapolis, MN) or eBioscience (San Diego, California); Antibodies to Mouse IL-1β and mouse caspase-1 were from R&D Systems or Santa Cruz Biotechnology (Santa Cruz, CA).
Wild type (WT) C57BL/6, caspase-1 KO and ob/ob (B6.Cg-Lep^ob/J) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). All mice were maintained at MUSC Hollings animal facility under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care & Use Committee (IACUC) at MUSC, and were conducted in accordance with federal regulations as well as institutional guidelines and regulations on animal studies.

Peripheral blood mononuclear cells at a density of 2 × 10⁶ cells/mL were treated with 100 ng/mL of the Toll-like receptor 4 (TLR4) ligand E. coli lipopolysaccharide (LPS; Invivogen, San Diego, CA) in the presence or absence of 1 mM SSZ (Sigma-Aldrich, St. Louis, MO), and incubated for 24 h at 37°C in 5% CO₂. The concentration of SSZ and the incubation time were selected after evaluation of cellular toxicity, where <1% of PBMC mortality [evaluated by Trypan blue exclusion staining and Fixable Viability Dye eFluor 506 (eBioscience)] was obtained at the chosen condition (n = 5, data not shown). Subsequently, the cells were stimulated with PMA and ionomycin (at 50 and 500 ng/mL, respectively), and incubated for 12 h at 37°C in 5% CO₂, all in the presence of 5 μg/mL of Brefeldin A and monensin, previous evaluation of cell viability with the Fixable Viability Dye eFluor 506. Finally, the cells were harvested, followed by surface and intracellular staining, as described above. In a fraction of individuals, PBMC were cultured with LPS plus SSZ in the presence or absence of the caspase-1 inhibitor Ac-YVAD-cmk or the pan-caspase inhibitor Z-VAD-FMK (at 0.25 and 5 μg/mL, respectively; both from InvivoGen), and the type of cell death was evaluated with the TACS Annexin V Kits (Trevigen), following the manufacturer's instructions.