Fla st

THP-1 cells (kindly gifted by Dr. Chang-Hee Suh, Ajou University, Medical Center, Suwon, Korea) were cultured in the RPMI 1640 medium (Gibco, BRL, Grand Island, NY, USA), supplemented with 1% of a penicillin/streptomycin solution (HyClone; Cytiva, Marlborough, MA, USA), and 10% of heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA, USA). THP-1 cells were differentiated into macrophages using 80 nM phorbol 12-myristate 13-acetate (PMA; Sigma–Aldrich Co., St. Louis, MO, USA) for 24 h. Human dermal fibroblasts (HDFs; ATCC, Manassas, VA, USA) and 293/hTLR5 cell line ((InvivoGen, San Diego, CA, USA) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco, BRL, Grand Island, NY, USA) containing 1% of the penicillin/streptomycin solution (HyClone; Cytiva, Marlborough, MA, USA), 10% of FBS (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 0.2% of Normocin (InvivoGen, San Diego, CA, USA). All the cells were incubated in a humidified atmosphere containing 5% of CO₂ at 37 °C (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Lipofectamine 2000 was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA); FLA-ST from InvivoGen (San Diego, CA, USA); and TH1020 from Tocris (Cookson, Bristol, UK). FLA-AA (flagellin from A. avenae) was purified according to a previously described protocol [62 (link)].