Ccl 191

A normal human lung fibroblast (NHLF) cell line (cat # CC2512) was purchased from Lonza (Lonza, Walkersville, MD, USA). We used 3 different batches of NHLF cells collected from 3 different donors as follows: Batch # 0000343490 was collected from a 24-year-old female; batch # 0000369145 was collected from a 43-year-old male and batch # 0000608197 was collected from a 67-year-old male. NHLFs were cultured in fibroblast growth medium (FGM-2) (Lonza), and cells at passage 3 to 7 were used for all experiments. Three human IPF lung fibroblast cell lines (CCL-134 and CCL-191) were purchased from American Type Culture Collection (ATCC) (ATCC, Manassas, VA, USA), and IPF lung fibroblasts (cat # CC-7231) purchased from Lonza at passage 2 to 5 were used for all experiments. IPF lung fibroblasts from ATCC were cultured in F12K medium, while those from Lonza were cultured in FGM-2 (Lonza). Early passages of IPF fibroblasts were used in this study as IPF-derived fibroblasts are known to display morphological features of replicative senescence even at early passage, unlike age-matched control fibroblasts at the same passage [30 (link), 31 (link)]. Human recombinant IFN-γ (Sigma-Aldrich, St. Louis, USA), PFD (TCI America, Montgomeryville, PA), TGF-β1 (Cell Signaling Technology, Danvers, USA) and PDGF-BB (R&D Systems, Minneapolis, USA) were used in our experiments.

Primary lung fibroblasts from IPF patients (CCL-191) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cell culture was performed according to the handling procedure of ATCC. Normal lung fibroblasts were isolated from healthy lung tissue of patients with lung adenocarcinoma (Table S1). All the samples were away from cancerous tissues. Primary lung fibroblasts from control or BLM-treated mice were isolated as previous report 22 (link). Under sterile conditions, human normal lung tissues and adult mouse lung from saline or BLM treated groups were scissored. We isolated the shallow surface tissues of lung to avoid vessels and bronchus. The tissues were minced into 5 mm³ pieces, and then placed in 75 cm² cell culture flasks. Ham's F12K medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Gibco) was added into the flasks 2 h later to prevent tissue blocks floating. After 3 days, when the cells isolated from tissues and reached 80% confluence, subculturing procedure was performed. Besides, cells were incubated with antibody against vimentin to determine the mesenchymal phenotype. All cells were cultured under 5% CO₂ at 37 °C and used for all experiments within 3-5 passages. Cells were synchronized for 6 h with 2% FBS F12K medium before directly being used for the subsequent experiments.

Primary cultures of human fibroblasts derived from lungs of IPF patients and healthy individuals were purchased from Carol Feghali-Bostwick (Medical University of South Carolina, Charleston, SC, USA). Informed consent was obtained under a protocol approved by the Institutional Review Board for Human Research at the Medical University of South Carolina. All patients fulfilled the criteria for the diagnosis of IPF as established by the American Thoracic Society and the European Respiratory Society [44 (link)]. Control specimens were collected during surgical resection of solitary pulmonary nodules, and lung cancer was excluded by pathology after surgery [45 (link)].
Cells from the IPF lung myofibroblast cell line CCL-191, the normal lung myofibroblast cell line CCL-151, and the human Jurkat T cell line were procured from American Type Culture Collection (ATCC, Manassas, VA, USA).
Human and mouse lung myofibroblasts and Jurkat cells were cultured in RPMI 1640 fibroblast culture medium (Merck Group, Darmstadt, Germany), and were incubated at 37 °C in 5% humidified CO₂. Cells were passaged two times per week by dissociating monolayers with a mild trypsin solution and were used between passages three and six.