Calu 3 htb 55

293 T (CRL-3216; American Type Culture Collection [ATCC], Manassass, VA), Calu-3 (HTB-55; ATCC), and Huh-7 (Cat0403; Japanese Collection of Research Bioresources) cells were used in this study. 293T-hACE2, 293T-ACE2-furin, 293T-ACE2-TMPRSS2, and 293T-ACE2-CTSL stably expressing cells were constructed by co-transfection of the pLV plasmids with pCMV-Δ8.91 and pMD2.G in 293 T cells and selected with blasticidin (15 µg/mL), hygromycin B (150 µg/mL), puromycin (2 µg/mL) or puromycin (2 µg/mL) respectively. Expression of ACE2, furin, TMPRSS2, or CTSL was determined using quantitative (Q) PCR and Western blotting (Figure S2). Cells were cultured with Dulbecco's modified Eagle medium (DMEM, high glucose; HyClone, Logan, UT). One hundred units per milliliter of penicillin-streptomycin solution (Gibco, Germany), 20 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES, Gibco), and 10% fetal bovine serum (FBS, Pansera ES; PAN-Biotech, Aidenbach, Germany) were added to the culture medium. Lipofectamine 2000 (Invitrogen, Waltham, MA) was used as transfection reagent. Primers used for QPCR identification are listed in Table S1.

A human hepatoma cell line (Huh7, PTA-4583) and a transformed liver cell line (Hep3B, HB-8064) were obtained from ATCC (Manassas, VA). The cells were cultured in growth medium [Dulbecco’s modified Eagle’s medium (DMEM; Sigma‒Aldrich: St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; GeminiBio, West Sacramento, CA), 0.1% nonessential amino acids (Gibco-BRL), 1% Glutamax-1 (Gibco-BRL, Grand Island, NY), and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Irwindale, CA)]. Human lung cancer cell lines [Calu3 (HTB-55) and H1299 (CRL-5803)] were obtained from ATCC. These cultured cells were maintained in Eagle’s minimum essential medium (Gibco-BRL) supplemented with 1% Glutamax-1, 1% P/S, and 10% FBS. CD133-positive/negative Huh7, Hep3B, and H1299 cells were isolated using CD133-conjugated magnetic microbeads (AC133, Cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany). Two rounds of magnetic separation were performed. CD133-positive cells were cultured in growth medium as described above. The isolation quality was controlled by flow cytometry with an antibody against a different CD133 epitope (Santa Cruz Biotechnology, Dallas, TX).