Supporting Information

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SI Methods

For stable knockdown of the AhR in HaCaT cells, shRNA sequences for AhR silencing and a nonsilencing control (sequences given below) were cloned into PacI-ClaI sites of the lentiviral pCL1P.THPC vector (P. Rio and H. Hanenberg, unpublished work) using standard shRNA procedures (1). In the lentiviral vector, the shRNA was expressed of the human H1 promotor and the SFFV U3 region used to drive expression of the pac gene. Clones containing the shRNA inserts underwent sequence verification. Recombinant lentiviral particles were generated by transfecting 5 mg of the vector plasmids, the helper plasmid pCD/NL-BH (kindly obtained from Jakop Reiser, New Orleans, LA) and the VSV-G plasmid (generous gift from Dirk Lindemann, Dresden, Germany) into HEK293T cells as described (2). Lentiviral supernatants used to infect HaCaT cells in the presence of 8 mg/ml protamine were titered on HT1080 human fibrosarcoma cells (DSMZ, Hannover, Germany). Transduced HT1080 and HaCaT cells were selected in 4 mg/ml neomycin (Gibco, Eggenstein, Germany) for 5 days and subsequently used for further analyses.

For cloning of the mAhR into the pEGFP-C1 vector (Clontech, Heidelberg, Germany), a 2,418-bp fragment was amplified from C57/Bl6 mouse liver cDNA by using 4 mM solutions of each primer (sequences given below). The PCR reaction contained 5 units/ml Taq polymerase (Boehringer, Ingelheim, Germany) and 1.25 mM dNTPs. Amplification was performed 10 min at 95°C, 30 cycles of 30 sec at 95°C, 30 sec at 61°C, 2 min 72°C, and a final extension for 7 min at 72°C. After digestion of the vector and the fragment with HindIII and SalI according to the manufacturer's instructions, the PCR product was ligated into the vector (at room temperature, overnight), transformed into competent cells [Escherichia coli XL 1-blue (Stratagene, La Jolla, CA)] and plated onto 30 mg/ml kanamycin (Sigma-Aldrich, Munich, Germany) -containing agar plates. Colonies were picked the next day, grown overnight in kanamycin-containing LB-broth, and minipreps were performed using the Miniprep kit (Qiagen, Hilden, Germany). Clones containing the AhR insert underwent sequence analyses.

HaCaTs were plated on chamber slides (BD, Heidelberg, Germany) at a density of 5 ´ 10⁴ cells per chamber. pEGFP-AhR was transfected into the cells by using FuGene 6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions. Twenty-four hours later, transfected cells were exposed to 100 mJ/cm² UVB or 30 J/cm² UVA. Forty minutes later, cells were fixed for 10 min with 4% paraformaldehyde and washed with PBS. After brief drying, slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA), covered with cover glass, and sealed with nail polish. The GFP-coupled AhR was visualized under a fluorescent microscope (Olympus, Hamburg, Germany), and photographs were taken with a ColorViewXS digital camera (Olympus).

HaCaT cells were irradiated with 100 mJ/cm² UVB in the presence or absence of MNF and in the presence or absence of Trp (see above), or cells were incubated with 1-100 nM FICZ. After indicated times, RNA was prepared with the RNeasy kit (Qiagen) according to the manufacturer's instructions. Reverse transcription was performed as follows: for cDNA synthesis 1 mg of total RNA, 2 mg of p(DT)₁₅ primer (Roche, Switzerland) and 5 mM solutions of each dNTP were dissolved in 20 ml of H₂O and heated for 5 min at 65°C. The samples were chilled, and 8 ml of 5× RT-buffer (250 mM Tris·HCl, 375 mM KCl, 15 mM MgCl₂), 10 mM DTT, 40U RNA guard and 400 units of M-MLV reverse transcriptase (Invitrogen, ^Karlsruhe, Germany) were added to a final volume of 40 ml. The samples were reverse transcribed at 37°C for 50 min, and the reaction was inactivated at 70°C for 15 min.

Real-time PCR was performed by using the LightCycler instrumentation (Roche). The PCR mix consisted of 1/10 volume of QuantiTect SYBR green PCR Master Mix (Qiagen, Hilden, Germany), 0.5 mM solutions of each primer, 2 ml of cDNA, and DEPC (diethylpyrocarbonat) -treated H₂O in a final volume of 20 ml. The application started with an initial incubation step of 15 min at 95°C to activate the DNA polymerase. The conditions for PCR amplifications were 40 cycles of 15 sec at 94°C for denaturation, 25 sec at 60°C of primer annealing, 30 sec at 72°C for elongation, and 2 sec at 72°C for fluorescence detection. PCR-primer sequences for human CYP1A1 and human COX-2 are given below. The quantification of PCR products was estimated from fragment-specific standard curves and was calculated with the LightCycler software 3. Standard curves were prepared by using 10² to 10⁶CYP1A1 or COX-2 cDNA copies per ml and amplified as described above.

To detect the native AhR in the nucleus after UVB irradiation, nuclear extracts were prepared. Cells were harvested 40 min after UVB and sham irradiation in PBS containing 1 mM Na₃VO₄ and 5 mM NaF. After washing with hypoton buffer including 20 mM Hepes, 20 mM NaF, 1 mM Na₃VO₄, 1 mM Na₄P₂O₇, 1 mM EDTA, 1 mM EGTA, 125 nM ocadaic acid, and Protease-Inhibitor-Mixture III (Calbiochem, Darmstadt, Germany), the lysate was homogenized. The pelleted nuclei were resuspended in High-Salt buffer (hypoton buffer, 2 mM NaCl and 20% glycerol) and again homogenized. After centrifugation, the supernatant contained the nuclear proteins.

Immunoprecipitation of the nuclear proteins was done by using the Protein G Immunoprecipitation kit from Sigma-Aldrich with an AhR antibody from Affinity BioReagents (Golden, CO) according to the manufacturer's instructions. Precipitated proteins underwent Western blot analysis.

For the determination of intracellular tryptophan concentration, 1 ´ 10⁷ HaCaT cells were depleted for tryptophan for 6 hours and optionally afterward incubated for 1 hour with 1 mM tryptophan. Cells were intensely washed with PBS, harvested in PBS, and pelleted by centrifugation. After determination of the weight of the cell pellet, 1 ml of water was added, and the samples were frozen in liquid nitrogen. Cells were then thawed repetitively, and 1 ml trichloroacedoacid (10%) was added, incubated for 10 min on ice, and centrifuged for 10 min to remove high molecular weight components. The supernatants underwent GC-MS-MS analyses after two steps of derivatization. For the methylation, 1 ml of each sample was evaporated with N₂ at 40°C and then dissolved in 1.5 ml of 1.5 M HCl/MeOH for 30 min at 65°C. The sample were dried under a N₂ stream and redissolved in 1.5 ml of trifluoracetic anhydride (TFA) and acetylated for 1 hour at room temperature. After evaporation, the dried residues were resuspended in 200 ml of ethylacetate, and 1 ml was injected into the GC/MS-MS. The GC conditions were: 30 m × 0.25 mm ID, 0.25 mm film J&W ^{(Folsom, CA)} DB5MS, flow rate 1 ml/min helium, temperature program 60°C (5 min)-7°C/min^-190°C(0 min)-15°C/min^-250°C (10 min), injector temperature was 250°C. The MS-MS (VARIAN Saturn 2000 GC-MS/MS) was done with electron impact/ionization (EI). The quantification ions (m/z) were 198, 178, 156, and 128.

Total RNA was prepared from dorsal skin by using 1 ml Tri Reagent (Sigma) according to manufacturer instructions. The RNA was subjected to RNase-free Dnase I (Qiagen) digestion for 30 min, extracted with phenol and chloroform isoamyl alcohol (24:1), followed by chloroform isoamyl alcohol reextraction and isopropanol precipitation. RT-PCR analyses were done as described by Vogel et al. (3) for Cox-2 and Abel et al. (4) for Cyp1a1. For the housekeeping gene HPRT-1 (sequences given below) conditions were as follows: 57°C annealing, 15 sec elongation, 35 cycles.

FW 5'-CGTTT-ACCTTC-AAACTTT-ATTCAA-GAGATA-AAGTTTG-AAGGTA-AACGT-TTTTGG-AAAT and RW 5'-CGA-TTT-CCA-AAA-ACG-TTT-ACCT-TCA-AACTT-TATCTC-TTGAA-TAAAG-TTT-GAA-GGTA-AAC-GAT.

FW 5' TAC-TTCC-ACCT-CAGT-TGGC-TTCA-AGAG-AGCC-AACTG-AGGT-GGAA-GTAT-TTTTG-GAAAT and RW 5' CGAT-TTCC-AAAA-ATAC-TTCC-ACCT-CAGT-TGGC-TCTC-TTGA-AGCC-AACT-GAGG-TGGA-AGT-AAT.

FW 5'-CACA-CACA-AGCTT-CGATGA-GCAGC-GGCGC-CAACATC-3' (containing a HindIII cleavage site (underlined)) and RW 5'-CA-CAC-AG-TCGAC-TCA-ACTC-TG-CAC-CTT-GCT-TAG-3' (containing a SalI cleavage site (underlined)).

PCR-Primers for human CYP1A1 were 5'-TAGAC-ACTG-ATCT-GGCTG-CAG for the forward and 5'-GGG-AAGG-CTCC-ATC-AGC-ATC for the reverse resulting in a 146 bp fragment. Primers for human COX-2 were 5'-CCC-TTG-GGT-GTCA-AAG-GTAA for the forward and 5'-AACT-GATG-CGT-GAA-GTG-CTG for the reverse resulting in a 143 bp fragment.

HPRT-1 (forward 5'-GCG-TCGT-GATTA-GCGAT-GATG-AAC-3' and reverse 5'-CCT-CCCA-TCTC-CTTC-ATGA-CAT-CT-3')

1. Wiznerowicz M, Trono D (2003) J Virol 77:8957-8961.

2. Leurs C, Jansen M, Pollok KE, Heinkelein M, Schmidt M, Wissler M, Lindemann D, Von Kalle C, Rethwilm A, Williams DA, et al. (2003) Hum Gene Ther 14:509-519.

3. Vogel C, Boerboom AM, Baechle C, El Bahay C, Kahl R, Degen GH, Abel J (2000) Carcinogenesis 21:2267-2274.

4. Abel J, Li W, Dohr O, Vogel C, Donat S (1996) Arch Toxicol 70:510-513.