2.6. Cell Culture, Transfection and Lentiviral Transduction

Human bronchial epithelial cells (16HBE cells) were used in our study [33]. These cells were cultured in Dulbecco's modified Eagle's medium (HyClone, SH30022.01) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO₂ atmosphere. Cells were plated at 1.5 × 10⁵–2 × 10⁵ per ml in 6-well plates in antibiotic-free medium and transfected the following day with siRNA using Lipofectamine 2000 according to the manufacturer's standard protocol. The infection media was changed 6–8 h after transfection. 16HBE cells (2 × 10⁵/ml) were transduced with lentivirus at a multiplicity of infection (MOI) of 5–10 in 8 μg/ml polybrene (Sigma-Aldrich, H9268). Viral supernatant was exchanged for fresh media 12 h after inoculation. To generate stable cDNA expressing cell lines, the infected cells were selected with 1 μg/ml puromycin. To explore the role of TGF-β3 in autophagy and MUC5AC hyper-expressions, the cells were treated with TGF-β3 at concentrations of 0, 2.5, 5, and 10 ng/ml for a certain time (see Figure Legends). To explore the role of autophagy in MUC5AC hyper-expressions, the cells were co-treated with 3-MA (or Baf A1), and the final concentrations in the culture medium were 5 μM (10 nM for Baf A1).