Plasmids
The plasmid pEYFP-AHR-C1 used by29 encodes the human AHR (start at Ala 11). All pEYFP-AHR variants, which carry mutations at certain amino acids (AHRH39G, AHRR40D, AHRS36G, AHRS36G.H39G.R40D, AHRL118E.A121D.L122E, AHRL50D.A79D.F82D and AHRH39G.L50D.A79D.F82D.L118E.A121D.L122E.) were generated by GenScript (GenScript Biotech, Leiden, Netherlands).
Cell culture
The human hepatoma cell line HepG2 was purchased from DSMZ (Braunschweig, Germany). HepG2 cells were grown in RPMI 1640. MCF-7∆AHR cells were cultured in DMEM. Both cell lines were maintained in 5% CO2 at 37 °C in culture medium containing 10% (v/v) FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine. All media components were purchased from Pan-Biotech (Aidenbach, Germany). AHR-deficient variant of MCF-7 cells were kindly provided by Dr. P. Tarnow37.
Transient transfection
HepG2 and MCF-7∆AHR cells were seeded either on 10-cm dishes, 6-well plates (Techno Plastic Products AG, Trasadingen, Switzerland), 96-well plates (Techno Plastic Products AG, Trasadingen, Switzerland), or on glass bottom dishes (In VitroScientific, Sunyvale, CA, USA). On the next day, cells on glass bottom dishes were transfected by using Xfect (Takara Bio Europe SAS, Saint-Germain-en-Laye, France) and an appropriate DNA amount according to the manufacturer’s instructions. Cells on 10-cm dishes or on multiwell plates were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and an appropriate DNA amount as stated by the manufacturer’s instructions. After 4 h incubation, transfection medium was removed and replaced by new RPMI or DMEM medium, respectively.
Reagents
β-naphthoflavone (BNF) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Munich, Germany). Leptomycin B (LMB) was acquired from Santa Cruz Biotechnology (Santa Cruz Biotechnology Inc., Texas, U.S.A). Indirubin (IND) was provided from Enzo Life Sciences (Enzo Life Sciences GmbH, Lörrach, Germany) and 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) from LGC Standards (LGC Standards GmbH, Wesel, Germany). All chemicals were purchased at the highest purity available.
RNA analysis
RNA was isolated form cells by using RNeasy Midi kit in conjunction with QIAshredder (QIAGEN GmbH, Hilden, Germany). The purity and the concentration of RNA were determined using a plate reader device (Infinite M200 PRO, Tecan Trading AG, Männedorf, Switzerland). Reverse transcription was performed with the high-capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA USA).
Quantitative PCR (qPCR) was performed on a 7500 Fast Real-Time PCR instrument using FAST SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Hypoxanthine–guanine phosphoribosyl transferase (HPRT) was used as reference gene.
The primer sequences are listed in Table 1.
Luciferase assay
Cells used for the luciferase assay were seeded in 96-well plates (Merck KGaA, Darmstadt, Germany) at a concentration of 4.5 × 104 and 150 µL per well. Cell were transfected with pGL4.26-XRE provided by Dr. P. Tarnow38 together with AHR plasmids (WT or mutants). 24 h after stimulation cells were washed with PBS and lysed with lysis-puffer (10 mM Tris 0.1 mM, 2 mM EDTA, 1% Triton™ X-100) for 15 min. 50 µl luciferin solution and 150 µl assay puffer (Gly-Gly puffer, DTT and ATP) were injected per well. Luminescence was measured in a Synergy Neo2 plate reader (BioTek, Bad Friedrichshall, Germany).
RNA-silencing
For RNA silencing, cells were transfected with 80 pmol of siRNA (sc-29734) or the control siRNA (sc-37007) (all from Santa Cruz Biotechnology, Heidelberg, Germany) by using Lipofectamine 2000 according to the manufacturer’s instructions. The expression of the ARNT gene was measured by using the ARNT primer (sc-29734-PR) provided from Santa Cruz Biotechnology.
On-line confocal microscopy
HepG2 and MCF-7∆AHR cells are seeded and transfected as described above. Cells were monitored by confocal microscope (LSM 700, Carl Zeiss Jena GmbH, Jena, Germany) twenty four hours post transfection. For live cell imaging, cells were maintained in buffered medium in 5% CO2 at 37 °C. Simultaneously with the treatments, the imagining was initiated at a rate of one picture per minute. Microscopic image acquisition was done by using ZEN 2012 black edition software (Carl Zeiss Jena GmbH). For data analysis ZEN 2012 blue edition was used (Carl Zeiss Jena GmbH). The nucleus and the whole cell were defined and outlined as a region of interest (ROI), for which the fluorescence intensity was extracted.
Statistics
Data were analysed and graphed using GraphPad Prism (Graph Pad, La Jolla, CA, USA). Statistical analysis was done using paired Dunnett’s multiple comparisons test, one-way or two-way ANOVA, * p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
