Respiratory Syncytial Virus Infection and UPR in hSAECs

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).