Raw blue cells

In cell culture experiments, we used RAW264.7 mouse monocyte/macrophage cell line (ECACC, Salisbury, UK; passage number: 8–15) and RAW-Blue™ cells (Invivogen, Toulouse France, passage number: 10–14). RAW264.7 and RAW-Blue™ cells were cultured in 5% CO₂ at 37 °C in endotoxin-tested Dulbecco’s Modified Eagle’s Medium (high glucose, 4.5 g/L, 2 mM l-glutamine; Corning, Corning Incorporated Costar, Corning, NY) supplemented with 10% FBS. For culturing RAW-Blue™ cells, we also added 100 µg/ml normocin and 200 µg/ml zeocin to the medium. The day before the experiments, cells were plated onto 24- or 96-well plates and cultured overnight. Then medium was replaced to fresh one and cells were induced by 0.1 µg/ml or 1 µg/ml lipopolysaccharide (LPS; E. coli, 0127:B8; Sigma-Aldrich, Budapest, Hungary). The freshly synthesised tetralone derivatives were dissolved in DMSO and applied in 20 µM concentration as a pre-treatment, added 30 min before LPS induction. To exclude the effects of vehicle, both CTRL and LPS-treated cells received the same amount of DMSO.

RAW-Blue cells (InvivoGen, San Diego, CA, USA) are murine RAW264.7 macrophages that stably express an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. One million RAW-Blue cells were plated on a 100-mm Falcon cell culture dish (Corning Inc., Corning, NY, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, from which extracellular vesicles were removed by centrifugation at 110,000× g for 24 h, and Penicillin-Streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in the presence or absence of 0–10,000 ng/mL LPS, 20 ng/mL TNFα, 20 ng/mL IFNγ (R&D Systems, Inc., Minneapolis, MN, USA), or 10 μM N-(4-aminobutyl)-5-chloronaphthalene-2-sulfonamide hydrochloride (W13) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) at 37 °C for 72 h in 5% CO₂/95% humidified air. Photographs of cells were taken using a microscope equipped with a DP73 camera (Olympus, Tokyo, Japan). Conditioned medium and cells were collected, and the number and viability of cells were assessed by the trypan blue dye exclusion assay using the Countess II FL Automated Cell Counter (Thermo Fisher Scientific). Collected cells were washed twice with phosphate-buffered saline (PBS) and stored as cell pellets at −80 °C.