Recombinant human cytokines

Antibodies against myeloperoxidase (A0398) were from Dako/Agilent (Mississauga, ON, Canada); antibodies against citrullinated histone H3 were from Abcam (Ab5103); phospho antibodies were from Cell Signaling (Beverly, MA, USA). Ficoll-Paque Plus was from GE Biosciences (Baie d'Urfé, Qc, Canada); endotoxin-free (< 2 pg/ml) RPMI 1640 was from Wisent (St-Bruno, Qc, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA). Actinomycin D, cycloheximide, N-formyl-methionyl-phenylalanine (fMLP), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma (St. Louis, MO, USA). Kinase inhibitors and fluorescent probes were all purchased through Cedarlane Labs (Mississauga, Canada). PlaNET reagents, fluorescent chromatin-binding polymers, were from Sunshine Antibodies (https://sunshineantibodies.com/planet-001.html).

Antibodies raised against STK isoforms and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against Syk, as well as all phospho antibodies, were from Cell Signaling (Beverly, MA, USA). Ficoll-Paque Plus was from GE Biosciences (Baie-d'Urfé, QC, Canada); endotoxin-free (<2 pg/ml) RPMI 1640 was from Wisent (St-Bruno, QC, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA), and UltraPure LPS (from E. coli 0111:B4) was from InvivoGen (San Diego, CA, USA). Dimethyl sulfoxide (DMSO), N-formyl-methionyl-phenylalanine (fMLP), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma-Aldrich (St. Louis, MO, USA). Diisopropyl fluorophosphate (DFP) was from Bioshop Inc. (Burlington, ON, Canada). The protease inhibitors, aprotinin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), leupeptin, and pepstatin A, were all from Roche (Laval, QC, Canada). Kinase inhibitors were all purchased through Cedarlane Labs (Mississauga, Canada). All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.

Recombinant human cytokines and anti-human IL-36γ were purchased from R&D Systems (Minneapolis, MN). Inhibitors of p42/44 MAPK (PD98059 and U0216) and inhibitor for p38 MAPK (SB203580) were purchased from Merck (Darmstadt, Germany). siRNA for NF-κB p65, c-Jun and a control siRNA were purchased from Santa Cruz (Santa Cruz, CA). Antibodies against phosphorylated and total p42/44 MAPK (ERK1/2), p38 MAPK, JNK1/2, GAPDH and laminin A/C were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against phosphorylated c-Jun, NF-κB p65, phosphorylated IκBα were purchased from Santa Cruz. All other reagents were purchased from Sigma Chemical Co. (St Louis, MO).

Antibodies against P-Akt (#4060), P-mTORC1 (#5536), P-S6K (# 9234), P-PLCγ2 (#3871), and β-actin (#4967) were from Cell Signaling (Beverly, MA, USA); a pan histone antibody was from Sigma (#MABE71). Ficoll-Paque Plus was from GE Biosciences (Baie d’Urfé, Qc, Canada); Dextran T500 from Pharmacosmos (Holbæk, Denmark); endotoxin-free (< 2 pg/ml) RPMI 1640 from Wisent (St-Bruno, Qc, Canada); poly-L-lysine from Peptides International (Louisville, KY, USA). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA. Dimethyl sulfoxide (DMSO), N-formyl-methionyl-phenylalanine (fMLF), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma-Aldrich (St. Louis, MO, USA). All inhibitors were purchased through Cedarlane Labs (Missisauga, Canada). ProLong Gold Antifade, Hoechst 33342, propidium iodide, and 16% paraformaldehyde (PFA) were purchased from Thermo Fisher (Missisauga, Canada). PlaNET reagents (fluorescent chromatin-binding polymers) are no longer available from Immune Biosolutions or other suppliers; we therefore employed a close equivalent – fluorescent, 50-nm carboxylate microspheres (# 16661-10) from Polysciences Inc. (Warrington, PA, USA). These microspheres are referred to as PlaNET reagents throughout this study. All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.

To test the direct impact of LPS and inflammatory cytokines on hepatocytes, HepG2 (human hepatoma cells) cells were cultured in modified DMEM for 24 h under the above conditions used for RAW264.7 cells. The cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Then, HepG2 cells at 1 × 10⁵ cells/well were treated with LPS (Sigma-Aldrich) or recombinant human cytokines (R & D Systems, Minneapolis, MN, USA) at 5 ng/mL under the above conditions for 24 h before measurement of supernatant cytokines by ELISA kits for human cytokines (R & D Systems, Minneapolis, MN, USA) and Western blot analysis of the cell lysate with antibodies against human EPOR and human β-actin (Cell-Signaling Technology) following the above protocol.

Antibodies against citrullinated histone H3 and H4 were from Abcam (ab176843, ab219406, ab219407, ab81797); phospho antibodies were all from Cell Signaling (Beverly, MA, USA). Ficoll-Paque Plus was from GE Biosciences (Baie d’Urfé, Qc, Canada); endotoxin-free (< 2 pg/ml) RPMI 1640 was from Wisent (St-Bruno, Qc, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA). Monosodium urate crystals (MSU) were from Cayman Chemical (Ann Arbour, MI, USA). N-formyl-methionyl-phenylalanine (fMLP) and phenylmethanesulphonyl fluoride (PMSF) were from Sigma (St. Louis, MO, USA). All inhibitors, antagonists, and fluorescent probes were purchased through Cedarlane Labs (Missisauga, Canada). PlaNET reagents (fluorescent chromatin-binding polymers) were from Immune Biosolutions (https://immunebiosolutions.com/en/pipeline/planet-reagents/).