Kira8

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)).

For chemical treatment, the primary midbrain neurons at 14 DIV were exposed to vehicle (dimethyl sulfoxide, DMSO), 10 nM thapsigargin (an ER stress inducer, Sigma-Aldrich, #T9033), 100 nM GSK2606414 (a PERK inhibitor, MedChemExpress, #HY-18072)^{66 (link)}, or 100 nM KIRA8 (an IRE1α inhibitor, MedChemExpress, #HY-114368)^{67 (link)} for 48 or 96 h. For growth factor deprivation, the primary midbrain neurons at 14 DIV were deprived of growth factor by lowering the content of N2/B27 supplement in the culture medium from 1× to 0.1× for 48 h. After chemical treatment or growth factor deprivation, neurons were fixed with 4% PFA/PBS and immunostained with TH antibody and secondary antibody. The number of TH-positive neurons on each coverslip was counted under the confocal microscope with a ×40 objective. Counters were blinded to the genotypes and treatments of the samples. The survival rate of DAergic neurons was calculated by dividing the number of TH-positive neurons on each coverslip by the number of TH-positive neurons on the control coverslip (neurons from Dctn1^LoxP/LoxP P0 pups treated with vehicle or cultured in normal medium)^{62 (link)}.

hSAECs are immortalized primary human small airway epithelial cells [65 (link)] from ATCC (PCS-301-010, at passage 2). hSAECs were grown in SAGM small airway epithelial cell growth medium (Lonza, Walkersville, MD, USA) and used at passage 4. hSAECs undergo growth factor-induced cell-state transition [66 (link)] and maintain RSV-induced genomic and proteomic signatures representative of primary cells [27 (link)]. The human RSV long strain was grown in Hep-2 cells, prepared by sucrose cushion purification, tittered by methylcellulose plaque assay [26 (link),67 (link)] and quick-frozen until use. The selective IRE1α RNAse inhibitor KIRA8 (MedChemExpress, South Brunswick Township, NJ, USA) [68 (link)] was applied to the cells 2 h prior to RSV infection.