Cell Culture and Virus Propagation

Cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Serum Plus II, MilliporeSigma), 10 mM HEPES, 50 U/mL penicillin-streptomycin (Gibco), and 0.05 mM β-mercaptoethanol. HCT-8, Calu-3, and VeroE6 cells were obtained from the American Type Culture Collection. 293T-ACE2 cells that overexpress ACE2 (28 (link)) were a kind gift from Dr. Jesse D. Bloom (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). HCoV-OC43 (NR-52725) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) and propagated in HCT-8 cells using medium containing 2% FBS. HCoV-OC43 was titered in HCT-8 cells using a focus-forming assay and used for infections at an MOI of 0.01, unless otherwise indicated. SARS-CoV-2 Isolate USA-WA1/2020 (NR-52281) was obtained through BEI Resources, NIAID, NIH; propagated in VeroE6 cells; titered in VeroE6 cells using a plaque-forming assay; and used for infections at an MOI of 0.01, unless otherwise indicated. Cells were treated with small-molecule IRE1α inhibitors for 2.5 hours prior or tBHP for 2 hours prior to infection.