Pta 4583

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).

The Homo sapiens HCC cell lines Huh‐7 and HEPG2 were obtained from ATCC (Cat: ATCC® HB‐8065™ and ATCC® PTA‐4583; Baltimore, MD, USA). HEPG2 and Huh‐7 cells were preserved in T‐25 flasks at 37 °C and 5% CO₂ in an adjusted mixture of Dulbecco’s modified Eagle’s medium from Lonza BioWhittaker™ (Verviers, Belgium) and 10% (v/v) FBS from Sigma‐Aldrich [21].
At concentrations of 100 μg·mL⁻¹ and 100 U·mL⁻¹, penicillin/streptomycin (Lonza BioWhittaker™) was used. PBS, pH 7.2, was obtained from Lonza BioWhittaker™, and 2.5% trypsin was obtained from Gibco™ Life Technologies Corporation (New York, NY, USA).