Hepg2

A549 cells were cultured in Minimum Essential Medium Eagle (MEM) with L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10% fetal calf serum, 1 mM pyruvate, 10 mM HEPES, 50 μg/mL penicillin, and 50 μg/mL streptomycin (all supplements from Life Technologies, Grand Island, NY, USA). MCF-7 cells were maintained in MEM alpha modification with L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum, 1 μg/mL insulin, 50 μg/mL penicillin, and 50 μg/mL streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). HepG2 were propagated in Dulbecco’s Modified Eagle’s Medium DMEM—a high glucose medium (Sigma-Aldrich, St. Louis, MO, USA)—supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, 10 µL/mL MEM non-essential amino acids, 50 µg/mL penicillin, and 50 µg/mL streptomycin (all reagents from Life Technologies, Grand Island, NY, USA). A549, MCF-7, and HepG2 cells were purchased from the European Collection of Cell Cultures (ECACC, Salisbury, UK). The cell cultures were maintained under standard cell culture conditions at 37 °C in a humidified incubator under an atmosphere of 5% CO₂ and 95% air. Cells were passaged every 2–3 days to obtain exponential growth. The cells were maintained in the culture for no more than 20 passages.

The human hepatocellular carcinoma cell line HepG2 and the human embryonic kidney cell line HEK-T were obtained from the European Collection of Cell Cultures (ECACC, Porton Down, UK). The cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Pan-Biotech GmbH, Aidenbach, Germany), supplemented with 10% fetal calf serum (Pan-Biotech GmbH, Aidenbach, Germany), 100 U/mL penicillin, and 100 g/mL streptomycin (PAA Laboratories GmbH, Pasching, Austria) at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were passaged every 2–4 days (80–90% confluence), and seeded at 100,000 cells/cm² and 50,000 cells/cm² respectively for HepG2 and for HEK-T cells in 96-well plates.

Human cells lines (A2780, SW626, SKOV3, A2780Cis, A2780ADR, A549, HEPG2, OE19, PC3, HCT116) were purchased from the European Collection of Cell Cultures (ECACC) and tested at regular intervals to confirm mycoplasma free status. Cells were grown as adherent monolayers using RPMI-1640 culture medium supplemented with 10% v/v fetal calf serum, 1% v/v penicillin/streptomycin antibiotics, and 1% v/v 2 mM glutamine. Cells were maintained using 25 or 75 cm² flasks at 37 °C in a humidified atmosphere containing 5% CO₂ and passaged at regular intervals using trypsin-EDTA upon reaching 80–90% confluence.

Human colon cancer cell line (HT-29) and human liver carcinoma cell line (HepG2) were obtained from European Collection of Cell Cultures (Porton Down, Salisbury, UK). Both cell lines were grown in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Cells were passaged weekly and sub-cultured at confluence. Before each experiment, cells at 80% confluence were starved in serum-free medium for 24 hours. Growth-arrested cultures, in medium without FBS, were treated according to the various experimental purposes.
HT-29 cells were stimulated with a combination of cytokines consisting of 10 ng/ml IL-1α, 20 ng/ml TNF-α and 60 ng/ml IFN-γ. Each cytokine was previously diluted in 1% BSA in PBS. HT-29 cells were pre-treated with Resv, 5-ASA or both for 1 hour before the exposure to the cytokines and then maintained with the inflammatory stimulus for different time periods, depending on the experiment.

Human Caucasian hepatocellular carcinoma cells HepG2 were purchased from European Collection of Cell Cultures (ECACC No. 85011430). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of fetal bovine serum, 100 U/ml streptomycin, 100 µg/ml penicillin, 4 mM L-glutamine, 1% non-essential amino acids, and 1 mM sodium pyruvate. Cells were maintained at 37°C and 5% CO2 in a humidified incubator.
Primary human hepatocytes used in this study were obtained from two sources [19] (link): (i) from multiorgan donor HH52 (female; 60 years); the use of liver cells of donor HH52 was approved by “Ethical committee at the Faculty Hospital Olomouc”,and it was in accordance with Transplantation law #285/2002 Sb; “Ethical committee at the Faculty Hospital Olomouc” waived the authors from obtaining consent from the next of kin, regarding human hepatocytes obtained from liver donor HH52. (ii) long-term human hepatocytes in monolayer Batch HEP220770 (female; 35 years), Batch HEP220774 (female; 66 years) were purchased from Biopredic International (Biopredic International, Rennes, France). Cells were cultured in serum-free medium. Cultures were maintained at 37°C and 5% CO2 in a humidified incubator.

Hepatocellular carcinoma cell lines (Huh-7 and HepG2) were obtained from the European Collection of Cell Cultures. The cells were cultured and nuclear proteins extracted as described in [14 (link)]. Probes with 25 bases flanking the SNP of interest were designed (two per SNP, one for each allele), biotinylated, and annealed to form double stranded oligonucleotides. Probe sequences are given in Supplementary Table  1, in Supplementary Material available online at https://doi.org/10.1155/2017/1096916. The probes were incubated with nuclear extract (with or without competitor) at 25°C for 50 minutes. Following this the reactions were run on a 6% polyacrylamide gel for 210 minutes at 120 V and thereafter transferred on to Hybond-N+ membrane using Southern transfer [15 (link)]. DNA-protein complexes were cross-linked to the membrane and visualised and using the Thermo Scientific Lightshift Chemiluminescent Nucleic Acid Detection Module (according to the manufacturer's instructions, Thermo Fisher Scientific, Waltham, MA, USA).

Four human tumor cell lines were used: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma) and HepG2 (hepatocellular carcinoma), as previously described by Barros et al. [23 (link)]. MCF-7, HeLa and HepG2 were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and NCI-H460 was kindly provided by the National Cancer Institute (NCI, Bethesda, MD, USA). Extracts were tested at a final concentration range from 400 to 1.56 μg/mL. RPMI-1640 medium containing 10% heat-inactivated FBS and 2 mM glutamine was used to routinely maintain the adherent cell cultures at 37 °C, in a humidified air incubator containing 5% CO₂. For the experiments, each cell line was placed at an appropriate density (1.0 × 10⁴ cells/well) into 96-well plates. The cell growth inhibition was measured using sulforhodamine B, results were expressed as GI₅₀ values and Ellipticine was used as a positive control.