TGF-β Signaling Modulation in Lung Cells

Primary human lung fibroblast (Cat# CC-2512) and small airway epithelial cells (SAEC) (Cat# CC-2547) were purchased from Lonza. Lung fibroblasts were cultured in FGM-2 Fibroblast Growth Medium (Cat# CC-3132), and SAEC were cultured in SABM Small Airway Epithelial Cell Growth Basal Medium (Cat# CC-3119). Cells were seeded into 6 well plates for the treatment of 2 ng/ml TGF-β with or without 20 μM GSK4112 (Cat#: 3663; TOCRIS) and SR8278 (Cat#: S9576 Sigma) for 2 days. Human Fetal Lung fibroblast (HFL-1, Cat#: CCL-153) and human bronchial epithelial (BEAS-2B, Cat#: CRL-9609) cells were purchased from the American Type Culture Collection (ATCC) and stored in liquid nitrogen. The cells were thawed and cultured in DMEM/F12K medium (Cat#:113-20033; Thermo Fisher Scientific) with 1% Penicillin-Streptomycin-Glutamine (Cat#: 103-78016; Thermo Fisher Scientific), and 10% FBS (Cat#: 10082147; Thermo Fisher Scientific) for HFL-1 and 1% Penicillin-Streptomycin-Glutamine, 5% FBS for BEAS-2B. Cells were maintained under 5% CO₂ and 95% humidity. Before treatment, HFL-1 cells were starved in serum-free DMEM/F12K medium for 12 h, and BEAS-2B cells were serum-deprived in DMEM/F12K medium with 1% FBS. Then, the cells were treated with 2 ng/ml TGF-β with or without 20 μM GSK4112 (Cat#: 3663; TOCRIS) and SR8278 (Cat#: S9576 Sigma) for 2 days. After treatment, the cells were either lysed for protein/RNA quantification or fixed with 4% paraformaldehyde for immunofluorescence staining.