Human HCC cell lines PLC/PRF/5 and Hep3B as well as hepatoblastoma-derived

HCC cell line HepG2 were grown in Roswell Parm Memorial Institute medium or Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. Human HCC cell line Huh7 cells were grown in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL of streptomycin, 1% L-glutamine, and 1× nonessential amino acid (NEAA) miture. Human umbilical vein endothelial cells (HUVECs), IWR-1 concentration isolated from human umbilical cord veins by collagenase treatment, were cultured in M199 supplemented with 20% FBS, 100 units/mL of penicillin, 100 μg/mL of streptomycin, 3 ng/mL of BFGF, and 5 units/mL of heparin. For hypoxic exposure, cells

were placed in a hypoxia chamber in an atmosphere consisting of 94.9% N2, 5% CO2, and 0.1% O2. Real-time polymerase chain reaction (PCR) amplification was performed by using the SYBR Green PCR MK-2206 nmr Master Mix (Invitrogen/Applied Biosystems, Carlsbad, CA) and the ABI Prism 7300 real-time PCR system (Applied Biosystems), according to the manufacturer’s medchemexpress instructions. Calculations, based on the 2 method,15 were performed by using the following equation: R (ratio) = 2. The integrity of the amplified DNA was confirmed by determining the melting temperature. Data are expressed as fold change of the treatment groups in relation to the controls and were normalized to the levels of glyceraldehyde 3-phosphate

dehydrogenase (GAPDH). The primer sequences, designed by Primer3 and UCSC In-Silico PCR, were as follows: CypB forward, 5′-AATTCC ATCGTGTAATCAAGGACTT-3′; CypB reverse, 5′-TCTTGACTGTCGTGATGAAGAACT-3′; HIF-1α forward, 5′-TGATGACCAGCAACTTGAGG-3′; HIF-1α reverse, 5′-TGGGGCATGGTAAAAGAAAG-3′; GAPDH forward, 5′-TGACCACAGTCCATGCCAT-3′; GAPDH reverse, 5′-TTCTAGACGGCAGGTCA GGT-3′. Reactive oxygen species (ROS) were measured by using 2′,7′-dichlorfluorescein-diacetate (DCF-DA). Cells were loaded with 10 μM of DCF-DA at 37°C for 30 minutes and resuspended in 1 mL of phosphate-buffered saline. Fluorescence was measured by a flow cytometer. Mean dichlorodihydrofluorescein (DCF) fluorescence intensity was measured with excitation at 488 nm and emission at 525 nm. The assay was conducted as described previously,16 with slight modifications.