Penicillin streptomycin

The human hepatoma cell line HepG2 (European Collection of Cell Cultures, ECACC No 85011430) was grown in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin, and 4 mM l-glutamine (cell culture media and supplementations were obtained from PAA Laboratories) maintained at 37 °C and 5% CO₂. Cells were seeded in culture medium with 10% FBS (or 10% dextran-coated charcoal treated FBS (DCC-FBS) for transfection assays) for 24 h. HepG2 cells were either placed on 60 mm-diameter plates (0.375 × 10⁶ cells/mL) for RNA extraction or on 24 well-plates (0.12 × 10⁶ cells/mL) for transfection assays and RNA extraction from transfected cells. Cells were treated with TCDD 1 nM (Promochem) and/or E2 10 nM (Sigma–Aldrich) dissolved in dimethyl sulfoxide (DMSO, max. 0.25%; Sigma–Aldrich) in complete phenol red-free DMEM with 0.5% FBS (or without FBS for transfection assays). Additionally, simultaneous treatments with the AhR antagonist α-naphthoflavone (α-NF, Sigma–Aldrich) or the pure anti-estrogen ZK 191 703 (kindly provided by Dr. Karl-Heinrich Fritzemeier, Bayer-Schering, Germany) were performed.