Hsaec

The following cell lines were utilized in this study pHLF (Lonza Biosciences), hSAEC (Lonza Biosciences), MEF (BNCC100518, ATCC), PC3 cells (CRL-1435™, ATCC), HaCat cells (AddexBio T0020001). Primary human lung fibroblast cells (pHLF) and human small airway epithelium cells (hSAECs) were cultured according to supplier’s details (Lonza, Basel, Switzerland), Small airway epithelial cell growth medium (PromoCell, C-21070), respectively. Ogg1^+/+ and Ogg1^−/− mouse fibroblast (MF) cells were kindly provided by Dr. Deborah E. Barnes (Imperial Cancer Research Fund, Clare Hall Labs, United Kingdom). MF cells were maintained in DMEM/Ham’s F-12 (3:1) supplemented with 10% fetal bovine serum (FBS), glutamine, penicillin (100 U), and streptomycin (100 µg/mL). PC3 cells (CRL-1435™) maintained in F-12K medium, supplemented with 10% FBS, glutamine, penicillin (100 U), and streptomycin (100 µg/mL). HaCat cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, glutamine, penicillin (100 U), and streptomycin (100 µg/mL). Cells were routinely tested for mycoplasma contamination. Ogg1 expression in Ogg1^+/+ and Ogg1^−/− MF cells were characterized by qRT-PCR. Exon 3-4F: 5′-TGGACCTCGACTCATTCAGC-3′; R: 5′-CTTCGAGGATGGCTTTGGCA-3′; Exon 5-6F: 5′-TATGGCAGATTGCCCATCGT-3′; R: 5′-CCAGCATAAGGTCCCCACAG-3′.

Immortalized primary human small airway epithelial cells hSAECs and type II transformed alveolar carcinoma (A549) cells were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA). hSAECs were grown in SAGM (Lonza) and A549 in DMEM/F12 (Gibco supplemented with 10% FBS) in a humidified 5% CO₂ environment (19 (link), 23 (link)). RSV Long strain was prepared by sucrose cushion ultracentrifugation and titered by methylcellulose plaque assay (19 (link)). hSAECs were infected for 24 h at a multiplicity of infection (MOI) of 1.0 prior to harvest. For pharmacological induction of the UPR, hSAECs were treated for indicated times with various standard concentrations of tunicamycin (TM, 0.5-1 μg/ml) or thapsigargin (Tg, 50-100 nM). The kinase-inhibiting RNase attenuator (KIRA)-8, a selective IRE1α RNase inhibitor, was directly added to the SAGM at a concentration of 10 μM (31 (link)). The reagent was from MedChemExpress (South Brunswick Township, NJ, USA).

The establishment of HSAEC_4T53RD cells has been described previously [10 (link)]. Briefly, normal HSAECs, purchased from Lonza (Walkersville, MD, USA), were malignantly transformed by the introduction of Cdk4, hTERT, a dominant negative p53 mutant, K-rasV12, and cyclin D1, using retroviral vectors. The cells were cultured on collagen-coated dishes in serum-free SAGM medium supplemented with growth factors supplied by the manufacturer (SAGM Bullet Kit; Lonza), and were maintained at 37°C in a low-oxygen environment (3% O₂ and 5% CO₂) in a humidified incubator. TIG-3 human lung fibroblasts were obtained from the Japanese Collection of Research Bioresources Cell Bank and were cultured in DMEM supplemented with 10% FBS at 37°C in a low-oxygen environment (3% O₂ and 5% CO₂) in a humidified incubator.