Cell Culture and Treatment

A human monocyte leukemia cell line (THP-1) was obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured at a density of 5×10⁵ cells/mL in RPMI 1640 (Gibco, USA) medium supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin solution (Gibco, USA) at 37°C in a 5% CO₂ incubator. THP-1 monocytes were cultured in 6-well plates treated with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, USA) for 24 h to transform into adherent macrophages.¹⁶

THP-1 cells were induced with 2% cigarette smoke extract (CSE) for 24 h.¹⁷ After cigarette smoke extract exposure, non-adherent cells were discarded and adherent cells were incubated with 500 μL cold PBS for 5 min, then harvested by scraping the adherent cells with a plastic scraper into 1.5 mL tubes before centrifugation at 427 RCF at 4°C. The supernatant was removed and the cell pellet was stored at –80°C for protein expression analysis.