The human breast carcinoma cell lines MCF-7, T-47D, SK-BR-3, ZR75-1 and MCF-10A were obtained from the ATCC (Manassas, VA, USA), while 184hTERT cells  were a generous gift of Dr. Calvin Roskelley. MCF-7 and T-47D cells were maintained as previously reported . ZR75-1 cells were maintained as per MCF-7/T-47Ds. SK-BR-3 cells were maintained in Dulbecco's modified Eagle's medium (Sigma, Oakville, Canada) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 μg/mL streptomycin (Sigma) and 100 units/mL penicillin (Sigma). MCF-10A cells were maintained in DMEM F12 with L-Glutamine (HyClone) supplemented with 5% horse serum (Invitrogen, Burlington, Canada), 20 ng/mL epidermal growth factor (Invitrogen), 10 μg/mL insulin (Sigma), 0.5 μg/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma), 100 units/mL penicillin and 100 μg/mL streptomycin. 184hTERT cells were maintained in Clonetics® MEBM medium supplemented with Clonetics® SingleQuot, 400 μg/mL G418 (BioShop, Burlington, Canada), 1 μg/mL transferrin (BD, Mississauga, Canada) and 1.25 μg/mL isoproterenol (Sigma). Cells were cultured in a humidified atmosphere at 37°C and 5% CO2 .
Creation of L6-pRL has been previously described . To create the FLAG-GABPα construct, the human GABPα gene was PCR amplified using pCAGGS-E4TF1-60 (obtained from Hiroshi Handa, ) as the template with the primers specified in Additional File 1. The GABPα PCR product was cloned into the pSCT-Gal vector using the restriction enzymes XbaI/HindIII. The pSCT-Gal-GABPα construct was then digested with HindIII, filled-in by Klenow, and then cut with XbaI. The p3×FLAG-CMV-10 vector (Sigma) was cut with BamHI, filled-in using Klenow, and digested with XbaI. The complete FLAG-GABPα construct was obtained by ligation of these two fragments. To generate the FLAG-GABPβ construct, the human GABPβ gene was PCR amplified using pCAGGs-E4TF1-53 (obtained from Hiroshi Handa, ) as the template with the primers specified in Additional File 1. The GABPβ PCR product was then cloned into the pMAL-c2 vector (New England Biolabs (NEB), Pickering, Canada) using the restriction enzymes XbaI/SalI. Isopropyl β-D-1-thiogalactopyranoside (IPTG)/Xgal colour screening was used to select positive clones. The GABPβ fragment was then cut out of the pMAL-c2 vector using SalI, filled in by Klenow, and subsequently digested using XbaI. The p3×FLAG-CMV-10 vector was prepared by first cutting with BamHI, followed by a Klenow fill-in reaction, and then digestion using XbaI. The final FLAG-GABPβ construct was generated by the ligation of these two fragments. pTRE-tight-GABPβ was prepared by digesting FLAG-GABPβ with SacI (partial) and XmaI and cloning the FLAG-tagged GABPβ sequence into pTRE-tight (Clontech, Mountain View, CA, USA).
To create the NRF-1 expression vector, pTRE-tight-GABPβ was digested with BglII and MluI to remove the GABPβ sequence, but retain the FLAG sequence. The human NRF-1 coding sequence was PCR amplified from pSG5-NRF-1 , a generous gift of RC Scarpulla, using the primers specified in Additional File 1. The PCR product was digested with BglII and MluI and cloned into pTRE-tight with the FLAG sequence to create pTRE-tight-NRF-1. The FLAG-tagged NRF-1 sequence was cut from pTRE-tight-NRF-1 using SacI and XbaI and cloned into p3×FLAG-CMV-10 vector to create the NRF-1 expression vector, p3×FLAG-NRF-1.
The GABPβ proximal promoter sequences were PCR amplified using the primers and templates specified in Additional File 1. The promoter regions were cloned into the pRL-null reporter plasmid (Promega, Madison, WI, USA) using the restriction sites indicated in Additional File 1. Gb-270 multimer was prepared by cloning double-stranded oligonucleotides comprised of 3 repeats of Gb-270 (sequence specified in Figure 6) with HindIII (5') and KpnI (3') overhangs into pRL-null containing a TATA box derived from the albumin gene and a G-free cassette.
The human NRF-1 coding sequence was PCR amplified from pSG5-NRF-1  using the primers specified in Additional File 1. The PCR product was digested and cloned into the BamHI and HindIII sites of pMAL-c2. The recombinant protein was expressed and purified according to the manufacturer's protocol. The purified protein was eluted with 10 mM maltose in nuclear dialysis buffer (10 mM HEPES pH 7.6, 0.1 mM EDTA, 40 mM KCl, 10% glycerol, 1 mM dithiothreitol, 1 mg/mL leupeptin, 1 mg/mL pepstatin, 0.1 mM phenylmethanesulphonylfluoride (PMSF), 1% aprotinin, 1 mM benzamidine).
Dual luciferase assay
Approximately 24 h prior to transfection, cells were plated in 12-well plates at 1 × 105 cells/well. Cells were transfected in triplicate using a total of 250 ng DNA per well with 0.75 μL/well FuGENE (Roche, Laval, Canada) according to the manufacturer's protocol. The specific amounts of material used per well were: 25 ng of the CMV-luc internal control, 25 ng of each expression vector or empty vector control, 50 ng of shRNA plasmid and a Renilla luciferase reporter vector up to a total of 250 ng. Approximately 48 h post-transfection, the cells were washed with phosphate buffered saline (PBS), lysed in 150 μL passive lysis buffer (Promega), and 20 μL of the cell lysates were assayed using the Dual-Luciferase® Reporter Assay System according to the manufacturer's instructions (Promega) with a EG&G Berthold microplate luminometer.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared as previously described  with the exception that nuclear proteins were not concentrated by (NH3)2SO4 precipitation, but were dialyzed against 10 mM HEPES pH 7.6, 40 mM KCl, 0.1 mM EDTA, and 10% glycerol. Nuclear extracts or recombinant protein (2-4 μg unless otherwise indicated) were combined with 32 P-labelled oligonucleotides (1 ng) in binding buffer (25 mM HEPES pH 7.6, 5 mM MgCl2, 34 mM KCl, 50 μg/mL poly dI:dC (Sigma), 0.5 mg/mL bovine serum albumin (BSA)). Binding reactions (20 μL final volume) were incubated on ice for 15 min prior to separation on a 6% acrylamide 0.25 × TBE non-denaturing gel. For competition assays, unlabelled oligonucleotide competitors were mixed with 32 P-labelled oligonucleotides in binding buffer prior to the addition of nuclear extracts. For the supershift assay, 2 μg anti-NRF-1 (ab34682, Abcam, Cambridge, MA, USA) or PBS (negative control) was incubated with 4 μg of nuclear extracts for 30 min on ice prior to the addition of 32 P-labelled oligonucleotide in binding buffer. Oligonucleotide sequences are given in Figures 6 and 9 (positive strand only). The sequence of RC4, an oligonucleotide containing the NRF-1 binding sequence from the rat cytochrome C promoter (nucleotide -173 to -147), has been previously reported .
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed with the ChIP-IT™ Express kit according to the manufacturer's instructions (Active Motif, Carlsbad, CA, USA). Each immunoprecipitation reaction contained chromatin from 1.5 × 106 cells, and 2 μg of antibody (or water as a negative control). The following antibodies were used: acetylated histone H3K9 (06-599, Upstate Biotechnology, Lake Placid, NY, USA), haemagglutinin (Y-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA), RNA polymerase II (Covance, Emeryville, CA, USA), histone deacetylase I (ab7028, Abcam), NRF-1 (ab34682, Abcam), and Oct-4 (ab19857, Abcam). PCR primers amplified the BRCA1 promoter from position -341 to +116 ((+) 5'-GATTGGGACCTCTTCTTACG and (-) 5'-TACCCAGAGCAGAGGGTGAA)) and the GABPβ promoter from position -358 to -178 ((+) 5'-CTCCTACCCACCGCAGAAC and (-) 5'-CCATTTCTAGCGCTTCAGCC). A water blank (no template) and the initial chromatin were also subjected to PCR amplification as controls.
For dual luciferase assays involving siRNA knockdown, MCF-7 cells were plated at 5 × 104 cells/well in 24-well plates approximately 24 h prior to transfection. Cells were transfected in triplicate with siRNA (100 ng/well), GABPβ promoter constructs (175 ng/well), and CMV-luc (25 ng/well) for normalization of transfection efficiency, using TransMessenger™ Transfection Reagent (Qiagen, Mississauga, Canada) according to the manufacturer's protocol. Approximately 48 h post-transfection, the cells were washed with PBS, lysed in 75 μL passive lysis buffer and 20 μL of the cell lysates were assayed using the Dual-Luciferase® Reporter Assay System according to the manufacturer's recommendations. For Western blots, MCF-7 cells were plated at 2.5 × 105 cells/well in 6-well plates approximately 24 h prior to transfection. Cells were transfected with siRNA (1 μg per well) using Santa Cruz Transfection Reagent (Santa Cruz Biotechnology) according to the manufacturer's protocol. Approximately 72 h post-transfection, cells were washed twice with PBS and lysed in 200 μL loading buffer (2.5% SDS, 25 mM Tris-HCl pH 6.8, 10% glycerol, 1% apropotin, 1 mM dithiothreitol, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 0.1 mM PMSF, 1 mM NaF, 1 mM sodium orthovanadate, 20 mM β-glycerophosphate). siRNA used: siGAPDH (siGENOME® GAPD Control siRNA, Thermo Scientific Dharmacon, Lafayette, CO, USA) and siNRF-1 (5'-CGUUAGAUGAAUAUACUACtt, Ambion, Austin, TX, USA) .
RNA was isolated using the Genelute Mammalian Total RNA Miniprep Kit (Sigma). cDNA was generated by reverse-transcribing 2.5 μg of RNA for 5 min at 70°C and then 1 h at 42°C in a reaction mix containing 1 × MMluV reaction buffer (Invitrogen), 1 μg pol(N)6 primer (Pharmacia), 0.5 mM dNTPs, 1 μL RNAse OUT (Invitrogen), and 1 μL MMluV-RTase enzyme (Invitrogen) made up to 50 μL with diethylpyrocarbonate (DEPC)-treated water. Primer pairs specific to each of the GABP subunits and GAPDH were then used to amplify 2 μL of each RT product. In addition to the RT product and 500 ng of each primer, the reactions contained 1 × Thermopol buffer (NEB), 0.25 mM dNTPs, 1 μL Vent (NEB) and DEPC-treated water up to a final volume of 50 μL. The PCR protocol consisted of 4 min at 98°C, 29-33 cycles of (30 sec at 98°C, 1 min at 55°C, 1 min at 72°C) followed by 4 min at 72°C. Loading buffer was added to each sample to a final concentration of 2.5% Ficoll, 0.025% bromophenol blue and 0.1 mM EDTA, and 10 μL of each sample was resolved on a 1.5% agarose gel. Primers are specified in Additional File 2.
RNA and RT products were prepared as described above. Quantitative RT-PCR reactions for BRCA1 (with TBP as an internal control) were performed using the SuperScript® III Platinum® One-Step Quantitative RT-PCR system (Invitrogen) with 500 ng RNA per reaction and LUX™ primers specified in Additional File 2 according to the manufacturer's instructions. The PCR protocol consisted of 1 cycle of (900 sec at 55°C and 120 sec at 95°C), followed by 40 cycles of (30 sec at 95°C, 30 sec at 55°C, 30 sec at 72°C). BRCA1 expression for each cell line was calculated relative to the results for the MCF-7 cell line using the Pfaffl method .
Quantitative RT-PCR reactions for GABPβ were performed using the QuantiTect SyBr Green PCR kit (Qiagen) with 2.5 μL of RT product as per the manufacturer's instructions. Primer pairs and annealing temperatures (Tm) are specified in Additional File 2. The PCR protocol consisted of 1 cycle of 900 sec at 95°C followed by 45 cycles of (15 sec at 95°C, 30 sec at Tm°C, 30 sec at 72°C). GABPβ expression for each cell line was calculated relative to the results for the 184hTERT cell line using the delta-delta Ct method presented by PE Applied Biosystems (Perkin Elmer, Forster City, CA, USA).
Preparation of whole cell lysates
For GABP subunit complementation assays, cells were plated as described for dual luciferase assays. Transfections were performed using 3 μL FuGENE transfection reagent and 1 μg of each expression plasmid per well (total of 2 μg DNA per well), as per the manufacturer's instructions. Forty-eight hours post-transfection, the cells were scraped using a rubber policeman and lysed using 50 μL/well modified RIPA buffer (50 mM Tris-HCL pH 7.4, 1% Igepal C630, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/mL each of aprotinin, leupeptin and pepstatin, 1 mM sodium orthovanadate, 1 mM NaF) for 15 min at 4°C. An equal amount of 2 × SDS-PAGE loading buffer was added to each lysate. To determine the endogenous BRCA1, GABPα and GABPβ protein levels, cells were grow to 60% confluence, scraped using a rubber policeman and lysed using modified RIPA buffer for 15 min at 4°C. An equal amount of 2 × SDS-PAGE loading buffer was added to each lysate.
Whole cell lysates were resolved on a SDS-polyacrylamide gel, transferred to a nitrocellulose or PVDF membrane, and probed with the appropriate antibody. Primary antibodies included: anti-BRCA1 (0P92, 1:500, Calbiochem, San Diego, CA, USA), anti-GABPα (H-180, 1:500, Santa Cruz Biotechnology), anti-GABPβ (H-265, 10 ×, 1:5000, Santa Cruz Biotechnology), anti-FLAG (M2, 1:1000, Sigma), anti-NRF-1 (M01, 1:500, Abnova, Taipei, Taiwan), anti-γ-tubulin (GTU-88, 1:5000, Sigma), and anti-TBP (1TBP18, 1:2000, Abcam). Secondary antibody detection was performed by chemiluminescence (Thermo Scientific/Fisher, Nepean, Canada).
For immunofluorescence analysis of proteins, cells were plated on coverslips 24 h prior to transfection, in 12-well plates at a density of 1 × 105 cells/mL. Transfections were performed using 0.75 μL of FuGENE transfection reagent and 125 ng of each expression plasmid per well (total of 250 ng DNA per well), as per the manufacturer's instructions. Cells were incubated at 37°C for 48 h at which time the media was aspirated and the wells washed with PBS. The cells were fixed at room temperature using 4% paraformaldehyde in PBS, aspirated, washed with PBS, and then permeablized at room temperature using 0.5% TritonX-100 in PBS. The cells were incubated in blocking buffer (3% BSA, 10% Normal Goat Serum, 0.1% Triton X-100, 0.1% Tween 20 in PBS) at room temperature for one hour, followed by primary antibody solution (1:200 dilution of antibody in PBS, 3% BSA) at room temperature for one hour in a humidified chamber, and then washed with PBS. All steps were performed in the dark from this point onward. The coverslips were incubated in secondary antibody solution (1:100 dilution of antibody in PBS, 3% BSA) for one hour in the humidified chamber, washed with PBS, and then the nuclei were stained for 10 minutes with Hoechst in PBS. The coverslips were given a final wash with PBS and then mounted onto slides using Permount Anti-fade mounting medium. Images were visualized on a Leica TCS SP2 Multi Photon confocal microscope. FITC was excited using a 488 nm laser and detected at 525 nm ± 20, and Hoescht was excited using a 2-photon laser at 780 nm and detected at 450 nm ± 20. The imaging software used was Image Pro Plus.