Mouse tgf β1

To assess cell proliferation in vitro, primary lung fibroblasts (2 × 10⁵ cells/well) were plated in a 6-well plate containing DMEM-10. After removing the culture medium, the cells were loaded for 20 min at 37°C with CellTrace carboxyfluorescein succinimidyl ester (CFSE; ThermoFisher Scientific) diluted in pre-warmed PBS (2.5 μM). The cells were washed twice with culture medium to remove excess CFSE and then resuspended in fresh, pre-warmed DMEM-10. The cells were then treated for 48 h with mouse TGF-β1 (5 ng/ml; R&D Systems). The cells were detached using 0.05% trypsin/EDTA and analyzed by flow cytometry. To evaluate differentiation of lung fibroblasts, primary cells were plated overnight at 37°C to allow attachment as a monolayer. Following 24 h of incubation in culture medium containing 2.5% serum, cells were treated with mouse TGF-β1 (5 ng/ml) or vehicle. After 48 h, cells were rinsed with DPBS and detached using 0.05% trypsin/EDTA. The cells were fixed for 30 min at RT with 2% PFA in PBS, permeabilized with 0.05% Triton X-100 in FACS buffer (PBS containing 0.1% BSA and 0.1% sodium azide), and analyzed for expression of α-SMA by flow cytometry.

Mouse TGF-β1 (R&D Systems, Minneapolis, MN) and Recombinant Mouse TNF-α (Biolegend, San Diego, CA) were used in this experiment. IEOs were seeded the day before in IEO culture medium containing 32 ng/ml TNF-α. Two days after the TNF-α treatment, the IEOs were simultaneously treated with 8 ng/ml TGF-β1 and 32 ng/ml TNF- α in IEO culture medium. For inhibition assays, the inhibitors were treated with 32 ng/ml TNF-α in IEOs. The concentrations of inhibitors used for inhibitor assays were as follows; 10 µM SB202190 (Sigma-Aldrich, St. Louis, MO), 20 µM PD98059 (Sigma-Aldrich), 10 µM QNZ (Sigma-Aldrich), 10 µM Glibenclamide (Sigma-Aldrich).

Mouse dermal fibroblasts (MDFs; ScienCell, Carlsbad, CA, USA) were plated in 100 mm culture dishes 1 day prior to transduction. 10 µg/mL of polybrene (Millipore) was treated before lentiviral particle infection. 2 × 10⁵ infectious units of virus (IFU) of mouse DR5 shRNA (m) lentivirus (Santa Cruz Biotechnology, Inc.) or control shRNA lentivirus (Santa Cruz Biotechnology, Inc.) was added to the cells and incubated for 48 h with or without mouse TGF-β1 (10 ng/mL; R&D Systems) for activation. Cells were either collected for analysis of knockdown or plated on to 96 well plates. Caspase 3/7 activity was measured after 3 h of TLY012 incubation.