Isolation and culture of ASMCs

ASMCs were obtained from the trachea and bronchi of smoke-exposed and control SD rats as previously described (18,19). Briefly, the trachea and main bronchi were removed and separated from the lung, bronchial vessels and connective tissue, then dissected into cubes (dimension, 1×1×1 mm). All segments were cultured with Gibco Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Guangzhou Whiga Technology Co., Ltd., Guangzhou, China) and 100 mg/ml streptomycin (Guangzhou Whiga Technology Co., Ltd.) in conditions of 37°C and 5% CO₂ for 5 days. Cells were grown to 80% confluence (observed under an inverted phase contrast microscope; Leica Microsystems Wetzlar GmbH, Wetzlar, Germany), the medium was removed, the explants were washed twice with Gibco Hank's Balanced Salt Solution (Thermo Fisher Scientific, Inc.) and trypsinized (in 1 ml of a 0.25% trypsin solution; Guangzhou Whiga Technology Co., Ltd.) for 1 min. Next, ASMCs were cultured in DMEM medium supplemented with penicillin and streptomycin at the above-mentioned concentrations and 10% FCS. The medium was replaced every 2 days. ASMCs were identified by the characteristic hill-and-valley appearance and α-smooth muscle actin immunocytochemical staining. Experiments were performed on freshly isolated ASMCs.