Tgf β1

To quantify changes in cell number, 1 × 10⁵ lung stromal cells were plated in DMEM supplemented with 10% calf serum and 1% antibiotic/antimycotic solution. For serum treatment experiments, 1 × 10⁵ lung stromal cells from LFD-fed mice were plated with DMEM+5% serum collected from LFD- or HFD-fed mice+1% antibiotic/antimycotic solution. To test responses to TGFβ1, lung stromal cells from LFD-fed mice were grown in 5% serum from LFD- or HFD-fed mice supplemented with 10 µM of SB431542 or DMSO control or in DMEM+0.5% calf serum supplemented with 5 ng/mL recombinant mouse TGFβ1 (Bio-Techne Corporation, 7666-MB, Minneapolis, MN, USA) or PBS vehicle control. Cells were fed with media supplemented with serum every 2 days. All cell number assays were plated in triplicate and counted after 6 days with a hemocytometer and then pelleted for RNA extraction. Latent TGFβ1 was activated prior to quantification, and total TGFβ1 was measured in serum from LFD- and HFD-fed mice diluted 1:50 in buffer using TGFβ1 DuoSet ELISA (Bio-Techne Corporation, DY1679, Minneapolis, MN, USA) in duplicate per the manufacturer’s instructions.

Before experiments, both cell types (hPASMC or hPAEC) were serum-starved for 24 h. Cells were then treated for 24 h in the absence or presence of either IL-1β (0.1–100 ng/mL, Bio-Techne, Lille, France) or H₂O₂ (0.1–50 µM, Alfa Aesar, Kandel, Germany). In some experiments, cells were also treated in the absence or presence of TGF-β1 (0.1–10 ng/mL, Bio-Techne). To investigate the role of TGF-β1 and of its TβRI receptor in the effect triggered by IL-1β or H₂O₂, these treatments were also applied with or without anti-TGF-β1 blocking antibodies (1 µg/mL, 30 min pre-treatment, Bio-Techne), or with or without an inhibitor of TβRI receptor kinase activity (SB525334; 1 µM, 30 min pre-treatment, Tocris Bioscience, Bristol, United Kingdom). To investigate TβRI-dependent signaling pathways involved, cells were also treated with IL-1β or H₂O₂ in the absence or presence of an inhibitor of p38 (SB203580; 2 µM, 45 min pre-treatment, Tocris Bioscience) or of Smad3 (SIS3; 10 µM, 45 min pre-treatment, Tocris Bioscience). In parallel, cells were treated with TGF-β1 (5 ng/mL) for 10, 30, or 60 min to investigate Smad3 and/or p38 phosphorylation. Finally, to confirm that TGF-β1 secretion was upstream of NGF secretion, cells were also pre-treated with anti-NGF blocking antibodies (1 µg/mL, 45 min pre-treatment, Millipore, Molsheim, France) before IL-1β or H₂O₂ treatment.

BxPC3.MUC1, Neo, and Y0 cells were serum starved 18 hours before plating for the invasion assay. 50,000 cells were plated as in the invasion assay protocol. Cells were either left untreated, treated with 10 ng/ml of TGF-β1 (Peprotech, Rocky Hill, NJ, USA), or the c-Src inhibitor PP2 (Tocris), or a combination of 10 ng/ml of TGF-β1 and PP2. The invasion assay was performed as described above.