Total RNA was isolated with RNAiso-plus reagent (Takara, Shiga, Japan) and cDNA was synthesised with the ImProm-IITM Reverse Transcriptase system (Promega, Madison, WI, USA). Product formation was monitored continuously during PCR using Sequence Detection System software (ver.
1.7; Applied Biosystems, Foster City, CA, USA). Accumulated PCR products were detected directly by monitoring an increase in SYBR® reporter dye fluorescence. The expression levels of CYP1A1, CYP1B1, AhR, Sp1, TDO, GR, Nrf2, Pgc-1, Nrf2, CHOP and DR5 mRNAs in metformin-treated cells were compared to those in control cells at each time point using the comparative cycle threshold (Ct)-method. The following primer sequences were used: human AhR forward, 5'-ACT CCA CTT CAG CCA CCA TC-3'; human AhR reverse, 5'-GTG CAC AGC TCT GCT TCA GT-3'; human CYP1A1 forward, 5'-CAA GAG GAG CTA GAC ACA GT-3';
human CYP1A1 reverse, 5'-AGC CTT TCA AAC TTG TGT CT-3'; human CYP1B1 forward, 5'-TTC GGC CAC TAC TCG GAG C-3'; human CYP1B1 reverse, 5'-AAG AAG TTG CGC ATC ATG CT-3'; human GR forward, 5'- GAA CTT CCC TGG TCG AAC AGT T-3'; human GR reverse, 5'-GAG CTG GAT GGA GGA GAG CTT-3'; human Sp1 forward, 5'-AAA CAT ATC
AAG ACC CAC CA-3'; human Sp1 reverse, 5'-ATA TTG GTG GTA ATA AGG GC-3'; human TDO forward, 5'-GGG AAC TAC CTG CAT TTG GA- 3'; human TDO reverse, 5'-GTG CAT CCG AGA AAC AAC CT-3'; human Pgc-1, forward, 5'-AAC AGC AGC AGA GAC AAA TGC ACC-3';
reverse, 5'-TGC AGT TCC AGA GAG TTC CAC ACT-3'; human Nrf2, forward, 5'-TAC TCC CAG GTT GCC CAC A-3'; reverse, 5'-CAT CTA CAA ACG GGA ATG TCT GC-3'; CHOP, forward, 5'-CAA CTG CAG AGA TGG CAG CT-3'; reverse, 5'-CTG ATG CTC CCA ATT GTT CA-3'; DR5, forward, 5'-GCC CCA CAA CAA AAG AGG TC-3'; reverse, 5'-GGA GGT CAT TCC AGT GAG TG-3'; human -actin forward, 5'-TGG CAC CCA GCA CAA TGA A-3'; human -actin reverse, 5'-CTA AGT CAT AGT CCG CCT AGA AGC A-3'. 18S rRNA, forward, 5'-GCT GGA ATT ACC GCG GCT-3'; and reverse, 5'-CGG CTA CCA CAT CCA AGG AA-3'. The quantity of each transcript was calculated as described in the instrument manual and normalised to the amount of -actin or 18S rRNA as a housekeeping gene.
For detection of miR-34a, total RNA was extracted from the cells using the miRNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Total RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen). Levels of miR-34a
expression were detected using the miScript SYBR Green PCR kit (Qiagen) with specific primers for miR-34a (Bioneer, Seoul, South Korea). Values for miR-34a expression were normalised using specific primers to RNU6B (Bioneer, Seoul, South Korea) as an endogenous reference RNA.
7. Luciferase and -galactosidase assays
Cells were transfected with 0.5 g of human CYP1B1-Luc vector, pXRE- Luc, pGL3-ARE-Luc, PPRE-Luc reporter vector and/or 0.2 g of pCMV-- gal per well using Lipofectamine™ 2000. At 6 h after transfection, fresh medium was added. Cells were pretreated with metformin (1–5 mM) for 1 h, followed by treating with 10 nM TCDD or 30 M tBHQ for 24 h and lysed.
The lysed cell preparations were then centrifuged (12,000 rpm, 10 min), and the supernatant was assayed for both luciferase and -galactosidase activity.
Luciferase activity was measured using the luciferase assay system (Promega, Madison, WI, USA) with a luminometer, according to the manufacturer’s instructions. The -galactosidase assay was carried out in 250 L of assay buffer containing 0.12 M Na2HPO4, 0.08 M NaH2PO4, 0.02 M KCl, 0.002 M MgCl2, 0.1 M -mercaptoethanol and 50 g o-nitrophenyl-
-galactoside. Luciferase activity was normalized to -galactosidase activity and expressed as the proportion of activity detected, relative to the vehicle
control.
8. Western blotting
Whole-cell lysates were prepared in lysis buffer (50 mM Tris-HCl [pH 7.4], 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) and protein concentrations determined at 595 nm using the Bio-Rad protein assay kit. The denatured protein samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on a 10%
polyacrylamide gel, and blotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk and then incubated with the appropriate primary antibodies, followed by a HRP-conjugated secondary antibody. The blots were visualised using an ECL Western blot kit according to the manufacturer’s protocol. To investigate multiple protein targets under the same treatment conditions, the blot was stripped and re-used. Equal sample loading was confirmed by measuring -actin levels for whole-cell lysates and lamin B1 for nuclear fractions. The integrated optical density for the protein band was calculated by Image-J software, and the values were normalised to house-keeping gene -actin or Lamin B1.
9. Preparation of nuclear and cytosolic extracts
Nuclear extracts were prepared with a commercial kit according to the manufacturer’s instructions (Active Motif, Carlsbad, CA, USA). All steps were performed on ice or at 4°C, unless stated otherwise. Protease inhibitors (10 g/mLaprotinin and 10 g/mL leupeptin) and reducing agents (1 mM dithiothreitol and 1 mM phenylmethylsulphonyl fluoride) were added to each buffer just prior to use. Briefly, cells were incubated in five volumes of hypotonic Buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl) on ice for 15 min and homogenized. Nuclei were recovered by centrifugation at 900 × g for 15 min, and the supernatant was collected as the cytoplasmic extract. The nuclei were washed once using a nuclei wash buffer (10 mM HEPES, pH 7.9, 0.2 mM MgCl2, 10 mM KCl) and extracted using Buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2) for 30 min on ice. Insoluble material was removed by centrifugation at 14 000 × g for 10 min, and the supernatant used as the nuclear extract.
10. Immunoprecipitation (IP)
For IP, MCF-7 cells were grown to 70% confluence and subsequently treated with metformin in fresh medium for 24 h. The cells were harvested and nuclear extracts prepared with a commercial kit according to the
manufacturer’s instructions. The nuclear fraction was pre-cleared with 30 L of protein G plus/ protein A agarose for 1 h at 4C. IP was performed at 4°C for 3 h by incubation of 1 g anti-Pgc-1 antibody or normal IgG with cell extracts containing 2-mg protein, followed by addition of 30 L of protein G plus/protein A agarose for another 2 h. Immunoprecipitates were collected and washed five times with lysis buffer, resuspended in SDS sample buffer and boiled for 5 min at 95°C. PPAR was detected in bound proteins by Western blotting.
11. Chromatin immunoprecipitation (ChIP)
A ChIP assay was performed using the EZ ChIP kit (Milipore, Billerica, MA, USA) according to manufacturer’s protocol. Briefly, cells were cross-linked with a formaldehyde solution, and the chromatin sheared by sonication to isolate DNA fragments that averaged 300–500 bp. An anti-PPAR antibody was added to aliquots of pre-cleared chromatin and incubated overnight.
Input samples were incubated with the negative-control IgG. The immune complexes were captured by incubation with protein G-agarose for 1 h at 4°C. After reversing the crosslinks, DNA samples from immunoprecipitates were isolated, and RT-PCR were performed using the following specific primers flanking the PPRE of Nrf2 with 35 cycles of PCR amplifications:
Nrf2, forward, 5'-CGA GAG CGC TGC CCT TAT TT-3'; and reverse, 5'- GGG GGA CCT AGA GGA GGT CT-3', and the control GAPDH primers (Millipore).
12. Small interfering RNA transfection
Small interfering RNA (siRNA) targeting AhR, AMPK1/2, Sirt1 and the siRNA transfection reagent were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DR5 siRNA was obtained from Bioneer Co. (Seoul, South Korea). Cells, grown to 50% confluence, were transfected with specific siRNA or a non-specific control siRNA according to the manufacturer’s instructions for 48 h prior to experiments.
13. Sp1, HO-1, Sirt1, Pgc-1 and PPAR overexpression
The Sp1 and Sirt1 expression vectors were a gift from Dr. Kwang Youl Lee (Chonnam National University, South Korea). The full length cDNA of human HO-1 cloned in a mammalian expression plasmid vector (HO-1 expression vector) and pCMV6-Neo empty vector were purchased from OriGene Technologies (Rockville, MD, USA). The Pgc-1expression vector was purchased from Addgene (Cambridge, MA, USA). The PPAR
expression vector was purchased from OriGene Technologies (Rockville,
MD, USA). Cells were transfected with Sp1, HO-1, Sirt1, Pgc-1, PPAR or the empty vectors using Lipofectamine™ 2000 in antibiotic-free medium and cultured for 48 h prior to experiments.
14. miR-34a inhibitor and mimic transfection
miR-34a inhibitor and miR-34a mimic oligonucleotides were chemically synthesized by Bioneer (Seoul, South Korea). MCF-7 cells were transfected with the 200 nM oligonucleotides using Lipofectamine™ 2000, according to the manufacturer’s protocol. One day after transfection, the cells were treated with 5 mM metformin or left untreated for an additional 24 h. The cells were harvested for subsequent experiments 48 h post-transfection.
15. Measurement of intracellular reactive oxygen species (ROS)
The fluorescent probe H2DCFDA was used to measure intracellular ROS generation in MCF-7 cells after treatment with metformin or H2O2. Briefly, the confluent MCF-7 cells in the 96-well plates were pre-incubated with 20
M H2DCFDA for 30 min at 37°C, followed by treating the cells with metformin (1–5 mM) or H2O2 (100 M) for 1 h. The fluorescence intensity was measured at excitation and emission wavelengths of 485 and 530 nm, respectively, using a fluorescence spectrophotometer. The generation of ROS
16. Statistical analysis
All experiments were repeated at least three times. The data are expressed as means ± SD. One-way analysis of variance (ANOVA) was used to determine the significance of differences between treatment groups. The Newman-Keuls test was used for multi-group comparisons. Statistical significance was accepted for p values of < 0.05.