OVCAR3 (human ovarian adenocarcinoma) cells were obtained from the National Cancer Institute (Bethesda, MD). A549 (human lung carcinoma) and T98G (human glioblastoma multiforme) were purchased from American Type Culture Collection (ATCC; Manassas, VA). OVCAR3, A549 and T98G cells were maintained in RPMI 1640 medium (Invitrogen; Carlsbad, CA), Kaighn’s Modification of Ham’s F-12 Medium (F-12K; ATCC) and Eagle’s Minimum Essential Medium (EMEM; ATCC), respectively. These media were adjusted to 10% FBS, 2 mM L-glutamine and antibiotics (100 μg/ml gentamicin for RPMI 1640, or 50 units/ml penicillin-G and 50 μg/ml streptomycin for F-12K and EMEM).
IFNs, TRAIL and antibodies
Human IFN-α2c was generated as previously described.17
Human recombinant IFN-γ was obtained from Genentech, Inc. (South San Francisco, CA). Recombinant human TRAIL was purchased from R&D Systems (Minneapolis, MN).
For flow cytometry analysis, anti-IFNAR1 mouse monoclonal antibody (gift from Dr. Michael G. Tovey, CNRS-UPR 9045, France) was used. In addition, anti-IFNAR2 mouse monoclonal antibody (PBL Biomedical Laboratories; Piscataway, NJ), anti-TRAIL-R1 mouse monoclonal antibody (R&D Systems), anti-TRAIL-R2 mouse monoclonal antibody (R&D Systems), mouse immunoglobulin G 1 (IgG 1) (BD Biosciences, San Jose, CA), mouse IgG2a (BD Biosciences), and mouse IgG2b (R&D Systems) were used for flow cytometry analysis. Mouse monoclonal primary antibodies were used for western blot analysis against Stat1 (N-terminus), Stat2, Stat3, Jak1, Tyk2, IRF9 (BD Biosciences). β-actin (rabbit polyclonal antibody; Cell Signaling, Danvers, MA) was used as a loading control. Horseradish-peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a secondary antibody.
Transfection of RNA interference (RNAi)
Duplex RNAi, 25 bp and 21 bp, were purchased from Invitrogen and Dharmacon (Lafayette, CO), respectively. Stealth™ RNAi Negative Control Low GC (Invitrogen) and ON-TARGETplus™ siCONTROL Non-targeting siRNA #1 (Dharmacon) were used as negative-RNAi(#1) and (#2), respectively. Sequences for specific RNAi (Stealth™ (Invitrogen) and ON-TARGETplus™ (Dharmacon)) are shown in . The RNAi oligos were mixed with Lipofectamine (Invitrogen) in OPTI-MEM medium (Invitrogen), and the mixture was added to the medium on cells. The cells were incubated for 4 h at 37°C then the medium was replaced with medium containing antibiotics.
Phase-contrast imaging of cells under a microscope were taken using ZoomBrowser EX software (Canon; Tokyo, Japan) before harvesting the cells for cell cycle analysis.
Flow cytometry analysis
Flow cytometry was preformed to analyze cell surface receptors and cell cycle. To detect cell surface receptors, cells were harvested with 5 mM EDTA in phosphate buffered saline (PBS) 48 h after transfection with RNAi then washed with fluorescence-activated cell sorting (FACS) solution (5% FBS and 0.1% sodium azide in RPMI 1640). The cells were incubated for 60 min in the FACS solution with anti-IFNAR1 antibody (mouse IgG1), anti-IFNAR2 antibody (mouse IgG2a), anti-TRAIL-R1 antibody (mouse IgG1), anti-TRAIL-R2 antibody (mouse IgG2b), or mouse IgG1/IgG2a/IgG2b (as a negative control) followed by R-Phycoerythrin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 30 min on ice. The samples were washed with the FACS solution then measured and analyzed using CELLQuest software in FACS Calibur (BD Biosciences).
For cell cycle analysis, treated or untreated cells were harvested at the indicated time points and fixed with 70% ethanol in PBS at −20°C. The sample cells were washed with PBS then resuspended with 5 μg/ml of RNase A (Roche Diagnostic Corporation; Indianapolis, IN) in PBS for 25 min at 37°C and subsequently stained with 25 μg/ml of propidium iodine (PI) in PBS for at least 20 min on ice. Finally, the DNA content was detected by CELLQuest software in FACS Calibur. The percentages of sub-G1, G0/G1, S, G2/M and aneuploid phases and were analyzed by FlowJo software (Tree Star, Inc., Ashland, OR).
Western blot analysis
Cells were lysed in NP40 buffer. The NP40 buffer consisted of 1% NP40, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 20 mM Tris-HCl pH 7.5, 1 mM phenylmethylsulfonyl fluoride (PMSF), protease and phosphatase inhibitor cocktails. The cell lysates were then centrifuged at 15,000 × g for 10 min at 4°C. Equivalent amounts of proteins were added to SDS sample buffer (New England Biolabs, Ipswich, MA) under reducing conditions and separated on Tris-Glycine gels (Invitrogen). Subsequently, these proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5–10% dry-milk (Bio-Rad) in tris-buffered saline (TBS) with Tween20 (TBST), then washed with TBST and incubated with primary antibodies followed by HRP-conjugated secondary antibodies. After several washings with TBST, proteins were detected using an enhanced chemiluminescent substrate (Thermo Scientific; Rockford, IL). In some cases, the polyvinylidene difluoride membranes were stripped using antibody stripping solution (Chemicon International, Millipore; Billerica, MA).
Quantification of western blot analysis
Kodak Molecular Imaging Software (Eastman Kodak Company, Rochester, NY) was used to quantify western blots. The sum of pixel values in a rectangular area containing the target protein band was obtained and the background pixel values were subtracted from the same area. Each value from the target protein was divided by the value of its respective β-actin from which the background pixel values were subtracted, and the ratios were obtained following comparison to its respective control (negative-RNAi or pUNO-mcs).
Approximately 6 × 103 cells per well were plated in 96-well plates. After overnight incubation at 37°C, the cells were transfected and/or treated as indicated followed by incubation with 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO) for 4 h. The stained living cells were solubilized with acidified isopropanol and the absorbance was measured at 570 nm. The absorbance corresponding to the living cells was calculated as ratios (%) compared to the absorbance of the respective control. The ratios were expressed as cell viability, and 50% inhibitory concentration (IC50) was also determined by the ratios.
pUNO-mcs (multi-cloning site) plasmid, containing a blasticidin resistance gene for the selection of stable clones in the human cell lines, was obtained from InvivoGen (San Diego, CA). pUNO-hIRF9 plasmid, containing an 1197 bp fragment of the human IRF9 open reading frame, was also obtained from InvivoGen. The bacteria E. coli transformed with pUNO-mcs or pUNO-hIRF9 plasmid were plated on blasticidin Luria-Bertani (LB) agar plates and a single colony from each plate was grown overnight at 37°C in Terrific Broth (TB) supplemented with blasticidin (InvivoGen). The E. coli strain GT110 and E. coli strain GT116 were used for the expression of pUNO-mcs plasmid and pUNO-hIRF9 plasmid, respectively. The plasmid DNAs were purified using QIAGEN Plasmid Kits (Qiagen Inc. Valencia, CA) and the quality of the preparation was verified by agarose gel analysis and restriction map (data not shown). These plasmids were transfected by using FuGENE HD (Roche Diagnostic Corporation; Indianapolis, IN) into A549 and T98G cells for 24 h, then these cells were maintained with medium containing 10 μg/ml of blasticidin (MP medicals; Solon, OH) to construct stable transfectants.
Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR)
qRT-PCR experiments were performed in 0.2 ml 96-well PCR plates using Brilliant SYBR Green QRT-PCR Master Mix Kit, 1-Step (Stratagene; La Jolla, CA). Total RNA was purified using RNeasy Kit (Qiagen). Each reaction well in the 96-well PCR plates contained a total volume of 25 μl: 12.5 μl 2X SYBR QRT-PCR Master Mix, 0.5 μl forward primer, 0.5 μl reverse primer, 0.3750 μl Reference Dye (1 μl + 499 μl H2
O), 0.0625 μl StrataScript RT/RNase Block Enzyme Mixture, 5 μl total RNA (20 ng starting material) and 6.0625 μl H2
O. Mx3000p QPCR system (Stratagene) was used to carry out reactions. Primers of 20 mer length were designed using Primer3 software18
and synthesized by Integrated DNA Technologies (IDT; Coralville, IA). Lyophilized primers were resuspended in TE (10 mM Tris pH 8.0, 1 mM EDTA) prior to use, and working aliquots of 20 nmol were made. Three-step cycling protocol was performed consisting of 1 cycle lasting 30 min at 50°C, followed by 1 cycle lasting 10 min at 95°C, followed by 45 cycles of: 30 seconds at 95°C, 1 min at 55°C and 30 seconds at 72°C. A dissociation curve was performed on the final amplified PCR products in order to determine if the primers successfully amplified product and to assess that only 1 product exists. In addition, a comparative quantitation strategy was employed via the MxPro QPCR software so that the relative fold change values for respective genes could be calculated across treated versus untreated samples. The primer sequences tested were interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) (forward, 5′-AAAAGCCCACATTTGAGGTG-3′; reverse, 5′-GAAATTCCTGAAACCGACCA-3′), IFIT3 (forward, 5′-GAACATGCTGACCAAGCAGA-3′; reverse, 5′-CAGTTGTGTCCACCCTTCCT-3′), and TRAIL (forward, 5′-TTCACAGTGCTCCTGCAGTC-3′; reverse, 5′-ACGGAGTTGCCACTTGACTT-3′).
Multiple sequence alignment
Multiple sequence alignment was performed between consensus ISRE (5′-AGTTTCNNTTTCNC/T-3′ or 5′-A/GNGAAANNGAAACT-3′)19
and human DNA sequences of representative ISGs. Those were examined manually and additionally using CLUSTAL W (http://workbench.sdsc.edu/
). The human DNA sequences examined were 401 bp (−300 to +101) containing the transcription start site (TSS: +1) positioned upstream from the first exon. The TSSs were determined by DataBase of Transcriptional Start Sites (DBTSS: http://dbtss.hgc.jp/
) or previous reports.20, 21
The DBTSS contains a collection of TSSs experimentally determined.22
It has been reported that ISREs in most IFN-α response genes are usually located within 200 bp upstream of the TSSs.19
However it is also reported that at least 52% of human genes based on Reference Sequence (RefSeq; National Center for Biotechnology Information (NCBI)) were subject to regulation by putative alternative promoters23
and the TSS positions were not always fixed but rather frequently fluctuated up to 50 bp on average.24
Therefore, a wide range search (−300 to +101) containing regions upstream and downstream of TSS was performed for the multiple sequence alignment.
Electrophoretic mobility shift assay (EMSA)
Cells with the indicated treatment were washed with cold PBS containing phosphatase inhibitors (Active Motif, Carlsbad, CA), then solution A (described as below) was added and incubated for 10 min at 4°C. The cells were harvested and centrifuged at 230 × g for 5 min at 4°C. The pellet was washed with solution B (described as below) followed by centrifugation at <1,000 × g for 5 min at 4°C. Subsequently, the pellet was resuspended with solution C (described as below) for 15 min on ice and centrifuged at 15,000 × g for 10 min at 4°C. The supernatant was used as nuclear extract. Compositions of the solutions used were as follows; solution A = 10 mM HEPES-NaOH pH 7.82, 10 mM KCl, 0.5 M sucrose, 0.1 mM EDTA, 0.1% NP40, 1 mM dithiothreitol (DTT), 1 mM PMSF, protease and phosphatase inhibitor cocktails (Thermo Scientific), solution B = 10 mM HEPES-NaOH pH 7.82, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, protease and phosphatase inhibitor cocktails, and solution C = 10 mM HEPES-NaOH pH 7.82, 500 mM NaCl, 0.1 mM EDTA, 0.1% NP40, 10% glycerol, 1 mM DTT, 1 mM PMSF, protease and phosphatase inhibitor cocktails.
Double-stranded oligonucleotide probes with or without biotinylation was synthesized by Integrated DNA Technologies (IDT). The sequence of the consensus ISRE probe was 5′-TCTAGCTTTAGTTTCACTTTCCC
CTTTCGGTTT-3′ from a previous report.25
The underlined sequence is the consensus ISRE motif previously reported.19, 25–28
Sequences of ISRE-like(#1) and ISRE-like(#2) probes from human TRAIL gene were 5′-AGAGGAGCTtcTTTCAGTTTCCC
TCCTTTCCAA-3′ (−149 to −117) and 5′-GCTTCTTTCAGTTTCCCTcctTT
CCAACGACTA-3′ (−143 to −111), respectively. The underlined sequences are the ISRE-like motifs and the nucleotides shown in lowercase are mismatched with the consensus ISRE motif. Three μg of nuclear extract was incubated for 20–30 min at room temperature with 0.1 μM biotin-labeled oligonucleotide probe in 10 μl of binding buffer (23 mM HEPES-NaOH pH 7.82, 160 mM NaCl, 5 mM MgCl2
, 0.53 mM EDTA, 8% glycerol, 0.13% NP40, 1.3 mM DTT, 1 μg of poly (dI·dC) (Thermo Scientific), 1 μg of salmon and herring sperm DNA (R&D Systems), respectively). For competitive assays, 25-fold molar excess of unlabeled oligonucleotide probes or poly (dI·dC) were added to the binding buffer before adding the biotin-labeled probe. In , μg of anti-Stat1 (sc-346 X), anti-Stat2 (sc-476 X), or anti-IRF9 (sc-10793 X) rabbit polyclonal antibody (Santa Cruz) was added to the binding reaction mixtures 20–30 min after adding the biotin-labeled probe, followed by incubation on ice for 1 h. The reaction mixtures were then electrophoresed and separated through 6% DNA retardation gel (Invitrogen) in 0.5× Tris-Borate-EDTA (TBE) buffer (Invitrogen) at a voltage of 100 V for 210–220 min at 4°C. The DNA-protein binding complexes were electrically transferred to a positively-charged nylon membrane (Thermo Scientific) at 380 mA for 40 min at 4°C, followed by cross-link using UV cross-linker (Stratagene). The biotinylated signals of the DNA-protein binding complex were visualized using a LightShift Chemiluminescent EMSA Kit (Thermo Scientific) and a LAS-3000 luminescent image analyzer (Fujifilm; Stamford, CT).
FIGURE 5 ISRE-like motifs of TRAIL bound to ISGF3 complex in response to IFN-α2c. All EMSA experiments used the biotinylated consensus ISRE probe. Asterisks (*) indicate the consensus ISRE-ISGF3 binding complex. A, Twenty nM of negative-RNAi(#1)-transfected (more ...)
FIGURE 2 Effects of RNAi in OVCAR3 cells. A, Flow cytometry analysis was performed to determine the inhibition of IFNAR1 or 2 expression levels and dose dependence 48 h after RNAi transfection. The expression ratios (%) were obtained by subtracting the ratios (more ...)
A two-tailed student’s t-test was performed. Differences were considered significant if the probability value (p-value) was less than 0.05. In the case of multiple comparisons, the Bonferroni correction was performed, and then corrected p-values were determined.