2.1 Antibodies and Reagents
Rabbit anti-human NF-κB p50 antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. PA, USA. Goat anti-mouse FITC-labeled antibody was purchased from Chemicon International, Inc. (Temecula, CA, USA). Mouse anti-human HIF-1α monoclonal antibody was purchased from R&D systems, Inc. (Minneapolis, Minnesota, USA). Deferoxamine mesylate salt (DFO) and hydroquinone were purchased from Sigma Corporation (St Louis, MO, USA). FluoSpheres NeutrAvidin™-labeled microspheres were purchased from Invitrogen (Carlsbad, California, USA).
2.2 DNA Probe Preparation
Five types of double-stranded oligonucleotides were used in this study. These were biotin-labeled double-stranded DNA (dsDNA) containing seven times HIF binding site (HBS), biotin-labeled dsDNA containing five times NF-κB binding site (NBS), non-biotin-labeled HBS (cHBS), non-biotin-labeled NBS (cNBS), and non-biotin-labeled dsDNA VP16 without HBS and NBS (VP16). The HBS probe and cHBS probe were prepared by PCR using the pCRII-C2-9 vector which was constructed by our lab and the following primer pairs: 5'-Biotin-GCATCAAGCTTGGTACCG-3' as the forward primer and 5'-AGCTATCGATATCTGCAGAATTCGG-3' as the reverse primer for the HBS probe; 5'-GCATCAAGCTTGGTACCG-3' as the forward primer and 5'-AGCTATCGATATCTGCAGAATTCGG-3' as the reverse primer for the cHBS probe. The NBS and cNBS probes were prepared by PCR using the pNF-κB-hrGFP vector (Stratagene Corporation, Cedar Creek, TX, USA) and the following primer pairs: 5'-Biotin-TCATGTCTGGATCCAAGCTA-3' as the forward primer and 5'-CCGGGGATCCATCTA GATTACCCTCTAGAGTCT-3' as the reverse primer for the NBS probe; 5'-TCATGTCTGGATCCAAGCTA-3' as the forward primer and 5'-CCGGGGATCCATCTA GATTACCCTCTAGAGTCT-3' as the reverse primer for the cNBS probe. The VP16 probe was prepared by PCR using the pCMV-ampR-linker-VP16 vector which was constructed by our lab and the primer pair below: 5'-TATCCTGCAGTCCGCGTACAGCCGCGCG-3' as the forward primer and 5'-TGACCTCGAGCTACCCACCGTACTCGTCAA-3' as the reverse primer for VP16 probe. The sequences of DNA probes are given below:
Biotin-gcatcaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcggcttgtt ggagtgtacgtgtgtgctcccccaggcattggttgttggagtgtacgtgtgtgctcccccaggcatggttgttgg agtgtacgtgtgtgctccccaggcatggttgttggagtgtacgtgtgtgctccccaggcatggttgttggagtg tacgtgtgtgctcccccaggcatggttgttggagtgtacgtgtgtgctccccaggcatggttgttggagtgtacgtgt gtgctcccccagacgtatatacgtatataagccgaattctgcagatatcgatagct (the bold sequences indicate the HIF binding sites).
Biotin-tcatgtctggatccaagctaggggactttccgcttggggactttccgctggggactttccgctggggactttccg ctggggactttccgcggagactctagagggtatataatggatccccgg (the bold sequences indicate the NF-κB binding sites).
The cHBS and cNBS probes were used for a specific competition test, and VP16 was used for a nonspecific competition test.
2.3 Cell Culture
The human cervical cancer HeLa cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml of penicillin/streptomycin, and 10% (w/v) of sodium hydrocarbonate. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2.
2.4 Drug Treatment
The iron-chelator desferrioxamine (DFO) induces HIF-1α in normoxia. DFO prevents HIF-1α from proteolysis by inhibiting the activity of iron-dependent prolyl hydroxylases [11
]. To activate HIF-1, HeLa cells were seeded (1 × 105
cells per well) in a 24-well plate and cultured in DMEM for 8 h. Cells were treated with 100 μM of DFO in triplicate and then incubated for 16 h at 37°C. Hydroquinone, a reactive metabolite of benzene, is known to inhibit NF-κB activity. The cells were also treated with 25 μM of hydroquinone in triplicate and then incubated for 16 h at 37°C. After the medium was aspirated from the wells, the cells were rinsed with phosphate-buffered saline (PBS) and lysed with lysis buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.5 mM magnesium chloride (MgCl2
), 10 mM potassium chloride (KCl), 1 mM DTT, 10% (v
) protease inhibitor cocktail, and 0.1% (v
) Nonidet P-40).
2.5 Nuclear Extract
After incubation with or without the target drugs, the HeLa cells were scraped into DMEM medium, washed in cold PBS, and centrifuged at 500 × g
for 5 min. Next, HeLa cells at a concentration of 2.5 × 107
cells/ml were lysed in lysis buffer (10 mM HEPES), 1.5 mM MgCl2
, 10 mM KCl, 1 mM DTT, 10% (v
) protease inhibitor cocktail, and 0.1% (v
) Nonidet P-40) by pipetting up and down. The lysed cells were centrifuged in a microcentrifuge tube at 16,000 × g
for 5 min at 4°C, and the cytosolic supernatant was removed. The nuclear pellet, consisting of 1 × 108
cells/ml, was dissolved in extraction buffer (20 mM HEPES, 1.5 mM MgCl2
, 0.42 M NaCl, 0.2 mM EDTA, 25% (v
) glycerol, 1 mM DTT, and 1% (v
) protease inhibitor cocktail), incubated for 30 min on ice, and centrifuged at 16,000 × g
for 5 min [12
]. The supernatants containing nuclear proteins were collected and stored at -80°C. The protein concentrations were determined by the Coomassie Plus (Bradford method) protein assay reagent (Pierce, Oud-Beijerland, the Netherlands) according to the manufacturer's instructions.
2.6 Development of the MIA-TF System
Exactly 1.5 × 10-11 nmol of FluoSpheres NeutrAvidin™-labeled microspheres were mixed with PEG to a final concentration of 25 mM to block the microspheres in each reaction. After incubation at room temperature for 30 min, the blocked microspheres were diluted to 100 μl with bovine serum albumin (BSA) buffer (1% BSA in PBS) and centrifuged at 9,300 × g for 5 min. The supernatant was decanted and discarded; the pellet was then resuspended with 10 μl PBS.
Biotin-labeled dsDNA (1.5 × 10-8 nmol), prepared as described in the DNA probe preparation section, was added to an appropriate concentration of nuclear extract. The final volume of the reaction was adjusted with extraction buffer to 30 μl and incubated at room temperature for 30 min. The same amount of non-biotin-labeled dsDNA was added at the same step. After 30 min of incubation, the prepared microspheres were added to each sample and shaken gently for 15 min at room temperature. The sample was washed with 500 μl of washing buffer (0.02% Tween-20 in PBS) and then centrifuged at 9,300 × g for 5 min. After carefully discarding the supernatant, 40 μl of the primary antibody was added to each tube and incubated at room temperature for 1 h. After centrifugation at 9,300 × g for 5 min, the supernatant was discarded. Next, 500 μl of washing buffer was added into the sample, and the sample was centrifuged once more to remove the supernatant. Then, 40 μl of the secondary antibody (2.5 μg/ml in 1% BSA buffer) was added to each tube and incubated at room temperature for another 1 h. After the washing steps, the sample was resuspended in 500 μl BSA buffer and was ready for flow cytometry (Becton Dickinson, Mountain View, CA, USA). The parameters for the measurement of fluorescence intensity were as follows:
2.7 Whole Cell Extract
HeLa cells were seeded (3,500 cells per well) in a 96-well plate. After incubation for 24 h, cells were scraped in medium, washed in cold PBS, and centrifuged at 500 × g for 5 min. The cells were extracted in 50 μl of whole cell extraction buffer (20 mM HEPES, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM MgCl2, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 10% (v/v) protease inhibitor cocktail) by pipetting up and down and incubated on ice for 20 min. After centrifugation at 3,700 rpm (Swing-bucket rotor A-2-DWP, Eppendorf, Hamburg, Germany) for 5 min, the whole cell extract was in the supernatant, the sample was analyzed by modified-MIA-TF after the protein concentrations of the whole cell extracts were determined by the Coomassie Plus (Bradford method) protein assay reagent (Pierce, Oud-Beijerland, the Netherlands) as specified in the instruction manual.
2.8 MIA-TF Directly Detect Transcription Factor in 96-Well Plates
Each well of 96-well microtiter plates was blocked with 300 μl of PBS buffer containing 5% BSA (BSA buffer) at room temperature for 1–2 h. Then, the BSA buffer in each well was discarded and the plate was washed three times with PBST buffer (0.05% Tween-20 in PBS buffer). MIA-TF was performed in the prepared 96-well microtiter plate. Briefly, the whole cell lysate was transferred into the wells of the blocked 96-well microtiter plate, and the blocked microspheres were added and mixed gently for 15 min. After centrifugation at 3,500 rpm for 5 min, the supernatant was carefully discarded and 40 μl of the primary antibody was added into each well and allowed to incubate at room temperature for 1 h. The wash procedure described above was repeated using centrifugation; 40 μl of the secondary antibody (2.5 μg/ml in 1% BSA buffer) was then added to each well and incubated at room temperature for 1 h. The samples were washed, resuspended in 200 μl BSA buffer, and finally their fluorescent activity was determined by flow cytometry.
2.9 Statistical Analysis
Data were analyzed using the SAS statistical software package (SAS Institute, Inc., Cary, NC, USA). The results were expressed as the mean ± SD. Differences in mean values were evaluated by a one-way ANOVA. The level of significance considered was p < 0.05.