Lps 055 b5

For TLR4 blocking experiments, 1 × 10⁵ human monocytes per well were plated in 24-well plates in 1 ml of medium and either left untreated or incubated with 1 μg/ml of a neutralizing IgG monoclonal antibody to human TLR4 (Invivogen, San Diego, CA, USA; Cat# mabg-htlr4) for 1 hour at 37 °C. Thereafter, the respective concentrations of E. coli LPS 055:B5 (Sigma-Aldrich) or Tth (purified from E. coli BL21 Star™ (DE3) were added. After another 24 h, supernatants were harvested and analysed by ELISA.

We isolated PBMCs from whole blood by Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density centrifugation (ρ = 1.077 g/L) as previously described [22 ]. Cells were cultured in 96-well plates in RPMI-1640 medium (Biolot, Russia) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10 mM HEPES buffer, 0.5 mM 2-mercaptoethanol, 80 mg/mL gentamicin, and 100 mg/mL benzylpenicillin for 24 h at 37°C with 5% CO₂ in the presence or absence of LPS (055:B5; Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 200 ng/mL.

Normal human hepatic cell line of L-02, purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), was cultured in Roswell Park Memorial Institute (RPMI) 1,640 medium (Gibco, Grand Island, NY, United States) containing 10% fetal bovine serum (Gibco), penicillin of 100 U/ml, and streptomycin of 100 μg/ml in a humidified incubator, with CO₂ at 5% and 37°C. The dose of the d-galactosamine (GalN)/lipopolysaccharide (LPS) model (D-GalN/LPS: 10 mM/10 ng/ml) was determined based on previous research Liu et al. (2018) (link) and our pre-experimental results. LPS (055: B5) and D-GalN (G1639) were obtained from Sigma-Aldrich Chemical Company (St. Louis MO, United States). In subsequent experiments, L-02 hepatocytes were divided into four groups: (a) Control group: Cells were cultured in RPMI 1640 medium alone. (b) D-GalN/LPS group: Cells were stimulated by D-GalN (10 mM)/LPS (10 ng/ml). (c) DEX + D-GalN/LPS groups: Cells were incubated with DEX (1, 10 μM) and D-GalN (10 mM)/LPS (10 ng/ml). (d) DEX + EX527+D-GalN/LPS groups: Cells were incubated with DEX (1, 10 μM), EX527 (10 μM), and D-GalN (10 mM)/LPS (10 ng/ml). According to the results of Cell Counting Kit-8 (CCK-8) assays (MedChemExpress, Monmouth Junction, NJ, United States), the concentration of DEX treatment in the subsequent experiments was determined as 1 μM.

LPS 055:B5 was purchased from Sigma (St. Louis, MO, USA). Mouse RIP3 antibody was from Abcam (ab56164). Mouse p-RIP3 antibody and MLKL antibody were obtained from Prof. Jiahuai Han’s Lab in Xiamen University, China[22 (link)]. The GAPDH antibody, XIAP, caspase-3, cleaved caspase-3, caspase-8, and cleaved caspase-8 antibody were purchased from Cell Signaling Technology (CST5174). Horseradish peroxidase-conjugated goat anti-rabbit antibody, bicinchoninic acid (BCA) protein assay kit and enhancer chemiluminescent (ECL) reagent were purchased from Pierce Biotechnology (Rockford, IL, USA). The protein extraction kit and TUNEL in situ apoptosis detection kit were from Key GEN Bio TECH (Nangjing, China). Propidium iodide (PI) was from Sigma (P4170).