Hacat human keratinocyte cells

HaCaT human keratinocyte cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium containing heat-inactivated 10% FBS and 1% penicillin/streptomycin (Thermo Fisher Scientific). For RNA isolation, the HaCaT cells (0.5 × 10⁶ cells/well, 6-well plate) were incubated with EEVP (400 μg/mL) or dexamethasone (20 μM) for 24 h and then incubated with or without TNF-α/IFN-γ (10 ng/mL) (Thermo Fisher Scientific). Cell cytotoxicity was determined by quantifying the relative formation of formazan in proportion to the live-cell number using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The cells (5 × 10⁴ cells/well) were seeded in 96-well plates, incubated overnight, and treated with several concentrations of EEVP (25–400 μg/mL) for 24 h. The cells were then incubated with the CCK-8 reagent for 1 h. The absorbance of the medium was measured using a microplate reader (450 nm; Molecular Devices, San Jose, CA, USA).