Pcs 300 030

HBEC-3KT (ATCC^® CRL-4051™) were cultured in the airway epithelial cells basal medium (ATCC^® PCS-300-030™) and supplemented with bronchial epithelial cells growth kit (ATCC^® PCS-300-040™), according to instructions from ATCC^®. HBECs were maintained at ~80% confluency to ensure epithelial morphology. HBECs were trypsinized with 1:3 dilution of 0.5% trypsin-EDTA (Invitrogen™, Life Technologies Inc, Burlington, ON, Canada) in PBS. The culture medium was changed to airway epithelial cells basal medium containing only 6 mM L-glutamine from the bronchial epithelial cells growth kit (and no other growth factors), to simulate a serum starvation condition 24 h prior to addition of the various stimulants.

Primary normal human lung fibroblasts (NHLFs) were purchased from Lonza and were maintained in manufacturer supplied growth medium (FGM-2 BulletKit, CC3132, Lonza). NHLFs were cultured up to six passages with media change every 3 days. Human primary SAEC (PCS-301-010, ATCC) were maintained in manufacturer supplied growth medium (PCS-300-030 and PCS-300-040, ATCC) supplemented with 100 U/ml penicillin, and 100 µg/ml streptomycin.

NSCLC cell lines were obtained from the US National Cancer Institute (Bethesda, MD, USA) (MTA 1-2702-09). All cells were incubated at 37 °C and maintained at 5% CO₂. H23, H226, IMR90 (normal lung fibroblast, ATCC CCL-186) and Lung Primary (Primary Small Airway Epithelial Cells; Normal, Human, ATCC PCS-301-010) cell lines were obtained from ATCC. IMR-90 cell was grown in DMEM/HIGH GLUCOSE medium (SH30243.01, Hyclone, Logan, UT, USA) containing 10% FBS. Lung primary cell was airway epithelial cell basal medium (PCS-300-030, ATCC, Manassas, VA, USA) with the bronchial epithelial cell growth kit (PCS-300-040, ATCC, Manassas, VA, USA).
NSCLC cells were grown in RPMI 1640 medium (Hyclone, Logan, UT, USA) plus 10% fetal bovine serum (FBS; Hyclone), penicillin and streptomycin. A small interfering RNA (siRNA) duplex targeting human GLS1, GOT2 and MDH2 (Santa Cruz, CA, USA) was introduced into the cells using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. As negative controls, cells were incubated with Lipofectamine 3000 (Invitrogen) and a negative siRNA (Santa Cruz). The hypoxic condition was achieved by incubating the cells in 1% of O₂, 94% of N₂ and 5% of CO₂ in a multigas incubator (Vision scientific. VS-9000GC, Seoul, Korea).

All NSCLC cell lines, were obtained from the U.S. National Cancer Institute (NCI; MTA no. 2702–09). Cells were incubated at 37°C and maintained at 5% CO₂. H23, H226, IMR-90 cell was grown in DMEM/HIGH GLUCOSE medium (SH30243.01, Hyclone, Logan, UT, USA) containing 10% FBS. Lung primary cell was airway epithelial cell basal medium (PCS-300-030, ATCC, Manassas, VA, USA) with the bronchial epithelial cell growth kit (PCS-300-040, ATCC, Masassas, VA, USA). NSCLC cells were grown in RPMI 1640 medium (SH30027.01, HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (SH30070.03HI, HyClone, Logan, UT, USA), penicillin, and streptomycin. siRNA duplexes targeting human ALDH1L1 (sc-78373), DHFR (sc-37078), GOT2 (sc-60052), and MDH2 (sc-89622) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were introduced into cells using Lipofectamine^® 3000 (L3000015, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. As negative controls, cells were incubated with Lipofectamine^® 3000 alone and a negative siRNA (sc-37007, sc-44230) (Santa Cruz). For ALDH overexpression, p3x FLAG-CMV-ALDH isoform constructs individually expressing ALDH1L1 and DHFR were produced by Cosmogenetech (Seoul, KOREA). Each cDNA sequence of ALDH1L1 and DHFR was obtained from NCBI. The plasmids were transfected into cells using Lipofectamine^® 3000 according to the manufacturer's instructions.

The human lung cancer cell lines A549 and H460 as well as mouse lung cancer cell line LLC were grown in Dubelco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 unites/ml penicillin, and 100 μg/ml streptomycin. The primary small airway epithelial cell (SAEC) was maintained in Airway Epithelia Cell Basal Medium (ATCC, PCS-300-030). All the cell lines were purchased from American Type Culture Collection (ATCC, USA) and cultured in a humidified incubator of 5% CO2 at 37 °C.

Viral kinetics in HRECs were performed by seeding 1 × 10⁵ cells per well in collagen-coated 24-well plates (Greiner Bio-one) using growth medium #2; for staining 96-well clear flat bottom black polystyrene surface-treated microplate (CellBIND Costar; Corning) was used, as described previously [15 (link)]. After 24 h of incubation, cells were washed twice with 500 μL of LHC base medium (Gibco, Thermo Fisher Scientific), and inoculated at MOI-2 and MOI-1 in triplicate with HCoV-NL63 or SARS-CoV-2 or mock inoculated with infection medium #2 (airway epithelial cell basal medium [PCS-300-030; ATCC] supplemented with 2% Ultroser G [Sartorius, Göttingen Germany], 1% MEM nonessential amino acids solution [Gibco, Thermo Fisher Scientific], 1% HEPES [Gibco, Thermo Fisher Scientific], 1% GlutaMax [Gibco, Thermo Fisher Scientific], 100 IU/mL penicillin, and 100 μg/mL streptomycin) and incubated at 37 °C with 5% CO₂ for 2 h. Cells were rinsed with LHC basal medium, fresh infection medium #2 was added to each well, and plates were incubated at 37 °C with 5% CO₂ for 96 h.