Crl 5803

The HCoV-229E strain (ATCC, VR-740) and HCoV-OC43 strain (ATCC, VR-1558) were purchased from ATCC, passaged once through MRC-5 cells (ATCC, CCL-171) and HCT-8 cells (ATCC, CCL-244), respectively, and were amplified in H1299 (ATCC, CRL-5803) or A549 cells (ATCC, CCL-185). Between 80 and 90% of the confluent cells were infected with HCoV-229E and HcoV-OC43 in a minimal volume of serum-free media for 2 h at 35 °C and 33 °C, respectively. Infected cells were incubated in media containing 2% FBS for 4 to 5 days. Culture supernatants were harvested when the CPE was observed, centrifuged at 4000 rpm for 5 min, passed through a 0.45 μm filter, and aliquoted for storage at 80 °C. We measured viral titer as the median tissue culture infective dose (TCID₅₀) per mL. The H1299 cells were seeded using 5 × 10³ cells per well in 96-well plates in 100 μL of maintenance media. After an overnight incubation, the cultured media was replaced with 100 μL of fresh media. Cells were incubated with 50 μL of diluted virus stock ranging from 10^–1 to 10^–7-fold. After incubation at 37 °C for another 7 days, the CPE was inspected under an inverted microscope to calculate TCID₅₀/mL.

In this experimental study, NIH3T3 mouse
embryonic fibroblasts (CRL1658, ATCC, USA) and
AML12 mouse hepatic cells (CRL-2254™, ATCC)
were cultured in Dulbecco’s Modified Eagle Medium
(DMEM, Invitrogen, USA) supplemented with 10%
fetal bovine serum (Sigma-Aldrich, USA) at 37˚C in
a humidified atmosphere of 5% CO2. The cells were
passaged every two days at 1:6 ratios. The cells were
treated with 500 nM Thapsigargin (Sigma-Aldrich,
USA) or 100 ng/ml Tunicamycin (Sigma-Aldrich,
USA) unless otherwise noted. Human lung cancer cell
line H1299 (CRL5803, ATCC), human ovarian cancer
line SKOV3 (HTB-77, ATCC), human ovarian surface
epithelial cell line HOSE (Beinachuanglian, PRC) and
human lung fibroblast HFL1 (CCL153, ATCC) cells
were cultured in RPMI 1640 (Invitrogen, USA) with
10% fetal bovine serum.

SUM159, HBL100, MCF7, MDA-MB-468, MDA-MB-435, MDA-MB-231, SKBR3, BT549, and 293T cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). All of the above cells grown in DMEM medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Hyclone Logan, UT, USA) and 1% penicillin/streptomycin (Life Technologies). A549, CRL-1848^TM, CRL-5803, CRL-5866, CRL-5833, 827, PC-9, 95D, HTB-182, and HTB-177 cells were obtained from the ATCC and maintained in RPMI-1640 (Hyclone) medium. The normal breast epithelial cell line MCF10A was purchased from ATCC and cultured in DMEM/F12 medium (Life Technologies) supplemented with 5% horse serum (Life Technologies), 10 μg/mL insulin (Sigma), 100 ng/mL cholera toxin (Sigma), 20 ng/mL epidermal growth factor (EGF, R&D), 0.5 μg/mL hydrocortisone (Sigma), and 1% penicillin/streptomycin. The normal lung epithelial cell line BEAS-2B was obtained from ATCC and maintained in LHC-8 (Gibco, USA). All cell lines were grown by incubation at 37°C in a humidified 5% CO₂ atmosphere.

Non-small cell lung carcinoma H1299 (ATCC CRL-5803™), hepatocellular carcinoma SK-HEP-1 (ATCC HTB-52™), and breast adenocarcinoma MCF-7 (ATCC HTB-22™) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). SK-HEP-1 cells were maintained in Dulbecco’s Modified Eagle’s Medium, while the other cell lines were cultured in Roswell Park Memorial Institute 1640 medium. All media were supplemented with 10% fetal bovine serum (Tico Europe, Amstelveen, Netherlands) and 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan). The cells were grown in a humidified environment with 5% CO₂ at 37 °C.

RAW-Blue™ cells (Mouse Macrophage Reporter Cell Line, InvivoGen, San Diego, CA, USA) derived from RAW 264.7 macrophages are grown in a culture medium containing Zeocin as the selectable marker. They stably express a secreted embryonic alkaline phosphatase (SEAP) gene inducible by NF-κB and AP-1 transcription factors. Upon stimulation, RAW-Blue™ cells activate NF-κB and AP-1, leading to the secretion of SEAP, which is detectable and measurable using QUANTI-Blue™, a SEAP detection medium (InvivoGen). RAW-Blue™ cells are resistant to Zeocin™ and G418 in the conditioned medium. NCI-H1299 (ATCC, CRL-5803™, carcinoma; non-small cell lung cancer) is an epithelial-like cell isolated from a White, 43-year-old male patient with carcinoma. A549 (ATCC, CCL-185™, lung carcinoma) is an epithelial-like lung cell isolated from the lung tissue of a White, 58-year-old male with lung cancer. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 0.1% plasmocin and grown in T-75 tissue culture flasks up to 70–80% confluence prior to experimental use.