Raw264.7 cell

Raw264.7 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, United States) and were cultured in PRIM-1640 (Thermo Fisher Scientific, MA, United States) containing 10% heat-inactivated fetal bovine serum (FBS; Corning, MA, United States) and 1% penicillin-streptomycin (PS). To evaluate the effect of oroxylin A on the TLR4/NF-κB signaling pathway, HEK-Blue cells were transfected with TLR4 and NF-κB SEAP reporter (Yang et al., 2021 (link)). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, CA, United States) supplemented with 10% (v/v) FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin. HEK-Blue hTLR4 cells were maintained with the intervention of 100 μg/ml Normocin (InvivoGen, No. ant-nr1) and 1× HEK-Blue Selection (InvivoGen, No. hb-sel). HEK-Blue null cells (TLR4 free) were grown with 100 μg/ml Normocin and 100 μg/ml Zeocin in the media. All cells were incubated in an incubator with a humidified atmosphere of 5% CO₂ at 37°C.

RAW264.7 cells and A549 cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in either arginine-free DMEM (Thermo Fisher Scientific Life Sciences, Waltham, MA, USA) or standard DMEM (containing 400 µM l-arginine; Sigma-Aldrich, St. Louis, MO, USA), both supplemented with fetal calf serum (10%; LabTech, Uckfield, United Kingdom). Confluent cultures were treated with LPS (RAW264.7 cells; 1 μg/ml; Sigma-Aldrich) or IL-1β (A549 cells; 10 ng/ml; R&D Systems, Minneapolis, MN, USA) for 24 h to induce iNOS and COX-2, in some cases in the presence of the NOS inhibitors, l-NAME (300 μM; Sigma-Aldrich) or ADMA (1 mM; Sigma-Aldrich). Increasing concentrations of ibuprofen arginate (Zambon Pharma), ibuprofen sodium (Sigma-Aldrich), l-arginine free base (Sigma-Aldrich), or the combination of ibuprofen sodium and l-arginine were also added such that the molar concentration of l-arginine present in each preparation was the same, or for ibuprofen sodium, the molar concentration of ibuprofen. After 24 h, media were collected for measurement of nitrite accumulation using the Griess reaction (Sigma-Aldrich) or PGE₂ using immunoassay (Cisbio, Codolet, France). Cell viability was assessed using the alamarBlue metabolic activity assay (Thermo Fisher Scientific).

RAW 264.7 cells and 3 T3-L1 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Dulbecco’s modification of Eagle’s medium (DMEM) was purchased from Gibco® by Life Technologies Co. (Carlsbad, CA) and Fetal bovine serum (FBS) from Hyclone (Logan, UT). Assay kits of total cholesterol, triglyceride, HDL-cholesterol, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Glutamic pyruvate transaminase (GPT), Gamma-glutamyl transferase (γ-GTP), Blood urea nitrogen (BUN), and Creatinine were purchased from Asan Pharm Co., Ltd (Whasung, Korea). (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dexamethasone (DEX), insulin, 1-methyl-3-isobutylxanthine (IBMX), Nicotinamide adenine dinucleotide phosphate (NADPH), Oil red O, Xanthine oxidase, and Hydrogen peroxide were purchased from Sigma Chemical Co. (St. Louis, MO).

RAW264.7 cells (murine macrophage cells) were obtained from American Type Culture Collection (ATCC). The cells were nurtured in folate-deficient DMEM medium containing 10% (v/v) FBS, 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C under an atmosphere of 5% CO₂ and 90% relative humidity. The medium was renewed every second day until 80% confluence was obtained. Before the experiments, once the precultured cells had reached 75% confluence, the cells were rinsed with PBS and trypsinized before harvest.

Mouse macrophage/monocyte RAW264.7 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and grown as previously described^{31 (link)} and details are provided in Supplemental Information (SI) Materials and Methods. The selective agonist for TLR2 [Pam₃Cys-Ser-(Lys)₄ hydrochloride (Pam3Cys; 10 μg/ml)] (Cat. #506350, Sigma-Aldrich, Saint Louis, MO, USA) was added to the cultures alone, or together with 3α,5α-THP (1.0 μM) in DMEM (without FBS and antibiotics) for 30 min and cells were harvested after 24 h. Selective agonists for TLR3 [polyinosinic–polycytidylic acid potassium salt (Poly(I:C); 25 μg/ml)] (Cat. #P9582, Sigma-Aldrich) or TLR7 [imiquimod (IMQ); 3 μg/ml] (Cat. #tlrl-imqs, InvivoGen, San Diego, CA, USA) were added to the cultures alone, or together with 3α,5α-THP (1.0 μM) in DMEM (without FBS and antibiotics) 24 h before cell collection.