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Human a549 lung carcinoma cell line

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The Human A549 lung carcinoma cell line is a well-established in vitro model derived from a human lung carcinoma. It is a commonly used cell line in research applications.

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6 protocols using human a549 lung carcinoma cell line

1

SARS-CoV-2 Spike Protein Inhibition Assay

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SARS-CoV-2 spike-pseudotyped lentiviral particles, VSV-G-pseudotyped particles and human ACE2-expressing HEK293 cells were purchased from GeneCopoeia, Rockville, MD. Recombinant COVID-19 Spike protein S1 was purchased from Abeomics, San Diego, CA. While anti-SARS-CoV-2 Spike S1 antibody was bought from BioVision (Milpitas, CA), anti-human ACE2 antibody was purchased from R&D Systems (Minneapolis, MN). Anti-human MyD88 antibody was bought from Millipore (Burlington, MA). Human A549 lung carcinoma cell line and F-12K medium were obtained from ATCC, Manassas, VA. Hank’s balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic mixture were bought from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals, Fort Collins, CO. ACE2:SARS-CoV-2 Spike Inhibitor Screening Assay Kit was purchased from BPS Bioscience, San Diego, CA. Anti-human IL-6 antibody and human IL-1β and IL-6 ELISA kits were bought from ThermoFisher, Waltham, MA.
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2

SARS-CoV-2 Spike Protein Inhibition Assay

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Human A549 lung carcinoma cell line (cat# CCL-185) and F-12K medium (cat# 30-2004) were purchased from ATCC. Hank’s balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic were purchased from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals. ACE2:SARS-CoV-2 Spike Inhibitor Screening Assay Kit (Cat # 79,936) was purchased from BPS Bioscience. Recombinant COVID-19 Spike protein S1 was purchased from MyBioSource (Cat# MBS553722) and Abeomics (Cat# MBS553722). Anti-SARS-CoV-2 Spike S1 antibody (Cat# A3000-50) was bought from BioVision. Human IL-1β ELISA and IL-6 ELISA kits were bought from ThermoFisher.
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3

Screening Inhibitors of SARS-CoV-2 Spike-ACE2 Interaction

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Eugenol, ursolic acid, oleanolic acid, and β-caryophylline were purchased from Sigma, St. Louis, MO. Methyl cellulose was bought from Spectrum Chemical, Gardena, CA. SARS-CoV-2 spike-pseudotyped lentiviral particles, VSV-G-pseudotyped particles and human ACE2-expressing HEK293 cells were purchased from GeneCopoeia, Rockville, MD. Recombinant COVID-19 Spike protein S1 (14-685) was purchased from Abeomics, San Diego, CA. Recombinant human ACE2 protein (18-739) was purchased from MyBiosource, San Diego, CA. Human A549 lung carcinoma cell line and F-12 K medium were obtained from ATCC, Manassas, VA. Hank’s balanced salt solution, 0.05 % trypsin, and antibiotic-antimycotic mixture were bought from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals, Fort Collins, CO. ACE2:SARS-CoV-2 Spike Inhibitor Screening Assay Kit was purchased from BPS Bioscience, San Diego, CA. Mouse TNFα ELISA kit was bought from ThermoFisher, Waltham, MA.
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4

Culturing A549 Cells and CIK Cells

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The A549 human lung carcinoma cell line (no. 59239596; American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). PBMCs from healthy adults were collected by cell apheresis (Fresenius Kabi Asia-Pacific, Ltd., Wanchai, China). CIK cells were generated by culturing PBMCs in RPMI-1640 supplemented with 10% FBS and containing 1,000 IU/ml recombinant IFN-γ (Shanghai Chemo Wanbang Biopharma Co., Ltd., Shanghai, China). At 24 h, 100 ng/ml anti-CD3 antibody (Wuhan Institute of Biological Products Co., Ltd., Wuhan, China), 1,000 IU/ml IL-2 (Four Rings Biotechnology, Beijing, China) and 1 ng/ml IL-1α (Invitrogen; Thermo Fisher Scientific, Inc.) were added. From day 5, the cells were replenished every 3 days with fresh medium containing 1,000 IU/ml IL-2. All cells were culturedat 37°Cin 5% CO2.
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5

Murine Embryonic Fibroblast Immune Signaling

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Murine embryonic fibroblasts (MEFs) were derived from wild type (WT) C57BL/6, TLR3/IPS-1−/− and TLR3/IPS-1/MDA5−/− mice and were maintained in α-MEM supplemented with 10% FBS, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin and 2mM L-glutamine. The A549 human lung carcinoma cell line (American Type Culture Collection) was maintained in α-MEM supplemented with 10% FBS. Vero cells (ATCC) were maintained in DMEM supplemented with 5% FBS. All cells were incubated at 37°C in a humidified 5% CO2 incubator.
Polyinosinic/polycytidylic acid (poly IC) was purchased from GE Healthcare (Buckinghamshire, UK). Doxycycline hyclate and oligomers were purchased from Sigma-Aldrich, Canada. Puromycin was purchased from Gibco (life technologies, USA). The PI3K inhibitor wortmannin (novex, life technologies) was reconstituted in DMSO. Human recombinant platelet-derived growth factor (PDGF-BB; Gibco) was reconstituted in PBS containing 0.1% BSA at a concentration of 50 ng/μl. Real-time PCR Taqman probes for human ISG56, ISG15 and GAPDH were purchased from Applied Biosystems (Streetsville, Canada).
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6

Cell Line Procurement and Culture

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Mouse Lewis Lung Carcinoma cells (LLC, CRL-1642), H1299 human lung carcinoma cell line (CRL-5803), H358 bronchioalveolar carcinoma cell line (CRL-5807) and A549 human lung carcinoma cell line (CCL-185) were purchased from the American Type Culture Collection. The cells were not specifically characterized for this study. DMEM, RPMI-1640 and Ham's F12 Nutrient Mixture were from Mediatech, Inc., (Manassas Herndon, VA). Fetal bovine serum (FBS) was purchased from Equitech-bio, Inc. (Kerrville, TX). Penicillin-streptomycin and trypsin-EDTA were from Invitrogen (Carlsbad, CA). Other chemicals were analytical grade.
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