Flagellin

ATP, Ultrapure LPS, colchicine, nocodazole, MDP, Phorbol 12-myristate 13-acetate (PMA), and doxycycline were obtained from Sigma. Imject Alum was obtained from Thermo. Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen. MSU crystals and cholesterol crystals were made as described46 (link)47 (link): cholesterol (Sigma) dissolved in 95% ethanol (12.5 g l⁻¹) was heated to 60 °C, filtered through filter paper and left at room temperature to allow crystallization; 8 g of uric acid (Sigma) was dissolved in 1,600 ml of boiling water containing 49 ml NaOH, and then PH was adjusted to 7.2, and crystals were formed by gradual cooling down. DOTAP (Roche), TransIT/LT1 (MirusBio), and TransIT/TKO (MirusBio) were used as transfection reagents. Mitotracker Red, Mitotracker Deep Red FM, TubulinTracker Green, Hoechst 33342, DAPI were obtained from Invitrogen. CytoTox 96 non-radioactive cytotoxicity assay (Promega) was used to measure lactate dehydrogenase (LDH) as an indication of cell viability following manufacturer’s instruction.

MKN45cl85 and 85As2 cell lines were established from the human gastric MKN-45 cancer cell line as described previously [7 (link)]. Cells were maintained in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 IU/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate (Nacalai Tesque, Inc.) in a 5% CO₂ atmosphere at 37°C. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Nacalai Tesque, Inc. LPS was purchased from Sigma Chemical (St. Louis, MO, USA). Flagellin was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). IRAK-1/4 inhibitor was purchased from Merck Millipore (Billerica, MA, USA).

Cells (1.5 × 10⁵) seeded in 12-well flat-bottom tissue culture plates were infected with bacteria at the indicated multiplicities of infection (MOI) in antibiotic-free culture medium for 2 h prior to washing and addition of complete culture medium. Alternatively, human glial cells were exposed to Pam3Cys, polyinosinic:polycytidylic acid (poly I:C) sodium salt, bacterial lipopolysaccharide (LPS), and/or Flagellin, ligands for TLR2, TLR3, TLR4, and TLR5, respectively. Pam3Cys was purchased from InvivoGen (San Diego, CA). Flagellin (isolated from Salmonella typhimurium strain 14028) was purchased from Enzolife sciences (Farmingdale, NY). LPS (isolate from Escherichia coli) and poly I:C were purchased from Sigma-Aldrich (St. Louis, MO). Following infection or exposure to bacterial products, cells were then cultured in the presence or absence of SP (Sigma-Aldrich) at a concentration of 5 or 10 nM. At the indicated time points, whole-cell protein isolates were collected and RNA was isolated for immunoblot analysis and semi-quantitative RT-PCR, respectively.