Immortalized hSAECs for RSV and UPR Studies

Immortalized primary hSAECs were obtained from American Type Culture Collection (ATCC, Gaithersburg, MD, USA) and grown in SAGM small airway epithelial cell growth medium (Lonza, Walkersville, MD, USA) in 5% CO₂ (11 (link),16 (link)). Sucrose cushion purified RSV Long strain was prepared and titered using methylcellulose plaque assay (11 (link)). hSAECs were infected at a multiplicity of infection (MOI) of 1.0 for 24 h prior to harvest. For induction of the UPR, hSAECs were treated for indicated times with various standardly used doses of 0.5–0.5 μg/ml tunicamycin (TM) or 50 nM thapsigargin (Tg). The selective IRE1α RNAse inhibitor KIRA8 (MedChemExpress, South Brunswick Township, NJ, USA) was added directly to the culture medium at a concentration of 10 μM where indicated (17 (link)).