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Supplementary Information
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p53-consensus DNA binding element coupled to luciferase was measured using the dual
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luciferase kit according to manufacturer’s instructions (Promega, Madison, WI). Briefly,
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HEK293T cells were transfected with p53wtluc or p53mutluc, p53-flag with or without si-
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angiogenin1, si-angiogenin2, or si-lamin while SAOS2 cells were transfected with p53wtluc or
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p53mutluc, p53-flag with or without increasing concentrations of angiogenin expression
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plasmids. Cells were harvested 48h post-transfection in 1x passive lysis buffer and luciferase
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assays were carried out in triplicate and normalized to renilla luciferase activity.
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Antibodies, reagents and growth factors. Rabbit polyclonal antibodies against human
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angiogenin, Bax, mouse monoclonal antibody against p53, Mdm2, p21, Bcl-2, myc and PARP
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were from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Anti-goat, anti-rabbit and anti-mouse
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antibodies linked to Alexa 488 or Alexa 594 and anti-fade DAPI (4’, 6’- diamidino-2-
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phenylindole) were from Molecular Probes, Eugene, OR.
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Angiogenin protein quantitation. The levels of angiogenin in the culture supernatant of cells
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were quantitated using a human angiogenin ELISA (ang-ELISA) (R&D Systems, Minneapolis,
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MN). Briefly, supernatants were collected from the cells, spun at 1,000 rpm for 10 min at 4 °C to
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remove the particulates and stored at -80 °C until use. Total soluble protein was quantitated by
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bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Since the concentration of protein
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present in the supernatant was the only variable between various cell types, an equal
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concentration of protein made up to 200 l was loaded onto each well, pre-treated with anti-
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angiogenin antibody followed by secondary antibody, developed using the substrate and read at
Supplementary Materials and Methods
Luciferase reporter assay. The effect of angiogenin expression and angiogenin silencing on the
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450 nm.
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Immunofluorescence assay (IFA). Cells in 8 well chamber slides were fixed with 4 %
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paraformaldehyde, permeabilized using 0.2 % Triton X-100, blocked with 5 % BSA in 1x PBS
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and stained with angiogenin and p53 antibodies for 1 h at 37°C. Cells were washed and
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incubated with an appropriate dilution of secondary antibodies conjugated with Alexa fluor 488
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and 594 respectively, for 1 h at RT. Stained cells were washed and viewed under a fluorescence
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microscope equipped with the Nikon Metamorph digital imaging system. Nuclei were visualized
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by using DAPI as a counter stain.
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Quantitative real-time RT-PCR. Two micrograms of total RNA harvested from cells was
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treated with DNase for 1 h and then reverse-transcribed into cDNA using random hexamers with
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high capacity cDNA reverse transcription mix for RT-PCR (Applied Biosystems, Foster City,
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CA). cDNA was used as a template with primers specific for: angiogenin, p21, p53, Bax and
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Bcl-2. Tubulin was used as internal control. PCR was performed using a Applied Biosystems
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7500 real-time PCR system with SYBR Green or Taqman PCR master mix (Applied
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Biosystems). The standard amplification program included 40 cycles of two steps each
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comprised of heating to 95°C and 60°C. Fluorescent product was detected at the last step of each
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cycle. The final mRNA levels of the genes studied were normalized using the comparative CT
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method.
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Western blotting. Lysates or immunoprecipitates were resolved by sodium dodecyl sulfate
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polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes,
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blocked with 5% skim milk and immunoblotted with antibodies against Bax, p53, Mdm2, p21,
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Bcl-2, myc, and PARP as well as anti-angiogenin antibody (Santa Cruz) for whole cell lysates.
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Immunoreactive bands were developed by enhanced chemiluminescence reaction (Pierce) and
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quantified following standard protocols.
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Fluorescent activated cell sorting (FACS): Cleaved caspase 3 assay samples were prepared as
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per manufacturer’s guidelines (BD Biosciences). The data were collected using a FACS Calibur
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flow cytometer (Becton Dickinson, Bedford, MA) and analyzed with FlowJoe software (Becton
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Dickinson) at the RFUMS flow cytometry core facility.
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Cell survival (MTT) assay. SAOS2 cells were transfected with p53-flag alone or in
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combination with increasing concentrations of angiogenin (0.5-5µg). At 48 hrs post-transfection,
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50 l of MTT reagent (Promega) was added to each well, incubated for 4 h and the absorbance
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was read at 570 nm. Cell viability was assessed according to the manufacturer’s protocol.
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Preparation of nuclear extracts. 293T cells transduced with si-Lamin, si-ang 1, si-ang 2, si-
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RL or transfected with ang-myc (5µg) were used to prepare nuclear extracts using a nuclear
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extraction kit (Active Motif Corp, Carlsbad, CA) as per the manufacturer's instructions. Protein
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concentrations were measured by bicinchoninic acid protein assay reagent (BCA: Pierce
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Biotechnology, Rockford, IL) and the extracts were stored at –80°C until used. Purity of the
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nuclear extracts was assessed by methods as described before (Sharma-Walia et al.2005,2011).
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Transcription factor activation assay. A total of 1µg of each nuclear extract was used for this
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assay. The level of active p53 transcription factor in the nuclear extracts was measured using an
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enzyme-linked immunosorbent assay (ELISA)-based TransAM p53 kit (Active Motif Corp.) per
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the manufacturer's instructions and by the methods described previously (Sharma-Walia et al.
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2005,2011). In this assay, transcription factors bind to the immobilized oligonucleotide
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containing the consensus sequences specific for the particular transcription factor, which is
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detected by a sandwich ELISA. The active form of the transcription factor contained in the
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nuclear extract specifically binds to this oligonucleotide mixture. The primary antibody used to
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detect the p53 transcription factor recognizes an epitope in the specific nuclear extract that is
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accessible only when these transcription factors are activated and bound to their target DNA. The
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detection limit for the TransAM p53 family kit is <0.5 µg of nuclear extract/well. A competition
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assay was done by premixing nuclear extracts for 30 min at 4°C with wild-type (WT) consensus
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(5´-GGACATGCCCGGGCATGTCC-3´) and mutated consensus oligonucleotides provided in
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the kit before addition to the probe immobilized on the plate.
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EMSA. p53 consensus oligonucleotide and p53 mutant oligonucleotide (identical to p53
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consensus oligonucleotides with the exception of a CA to TG substitution in the binding motif;
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5'-TAC AGA ACA TGT CTA AGC ATG CTG GGG-3') were obtained from Santa Cruz
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Biotechnology, Inc. The double-stranded oligonucleotides were labeled at the 5′ end with [γ-32P]
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ATP (American Radiolabeled Chemicals, Inc) using T4 polynucleotide kinase (Gibco BRL).
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Binding reactions were incubated on ice for 20 min and were performed in a 20-μl reaction
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volume containing 50 mmol/liter NaCl, 10 mmol/liter Tris-HCl, pH 7.5, 1 mmol/liter MgCl2, 0.5
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mmol/liter EDTA, 0.5 mmol/liter dithiothreitol, 9% (vol/vol) glycerol, 1 μg of poly(dI-dC), 5 μg
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of nuclear extract, and labeled probe (10,000 cpm). The resulting DNA-protein complexes were
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then size fractionated from the free DNA probe by electrophoresis at 200 V on a 6% native
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polyacrylamide gel (acryl/bisacrylamide ratio of 29/1) containing 2.5% glycerol, 50 mmol/liter
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Tris-HCl, pH 8.5, 0.4 mol/liter glycine, and 2.7 mmol/liter EDTA. These gels were dried at 80°C
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for 40 min, exposed to Kodak X-ray film, and visualized with a PhosphorImager (Molecular
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Dynamics), and the band intensities were calculated. A competition EMSA was performed by
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premixing nuclear extracts for 30 min at 4°C with WT-consensus and mutated oligonucleotides.
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Statistical analysis. In Fig. 1, 2 and 3, each reaction was done in triplicate and each bar
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represents the mean ± SD from three independent experiments. The statistical significance (t-
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test) was calculated with respect to control cells for each time point. In figures 1 and 2, fold
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change in gene expression was calculated by considering gene expression in untreated as 1. (*)
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p<0.01, (**) p<0.001, (***) p<0.0001.
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References:
Sharma-Walia N, George Paul A, Patel K, Chandran K, Ahmad W,Chandran B et al. (2011).
NFAT and CREB regulate Kaposi’s sarcoma-associated herpesvirus-induced cyclooxygenase 2
(COX-2). J Virol 84: 12733–12753.
Sharma-Walia N, Krishnan HH, Naranatt PP, Zeng L, Smith MS,Chandran B et al. (2005).
ERK1/2 and MEK1/2 induced by Kaposi’s sarcoma-associated herpesvirus (human herpesvirus
8) early during infection of target cells are essential for expression of viral genes and for
establishment of infection. J Virol 79: 10308–10329.
Supplementary figures
Figure S1. Angiogenin expression in HMVEC-d, SAOS2 and 293T cells. (A) cDNA prepared
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from the indicated cells was used for measuring angiogenin gene expression by quantitative real-
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time PCR. Each bar represents the mean ± SD from three independent experiments. (***)
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p<0.0001. (B) Supernatants harvested from the indicated cells were used for measuring
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angiogenin secretion by angiogenin ELISA. Mean ± SD of three independent experiments are
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shown. (***) p<0.0001.
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Figure S2. Expression of p53 in SAOS2 cells. SAOS2 cells were left untransfected or
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transfected with si-RL, si-ang1 or si-ang2 and Western blotted for p53. The blots were stripped
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and blotted for actin.
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Figure S3. Angiogenin treatment increases p53 expressing SAOS2 cell survival. SAOS2
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cells were transfected with the indicated plasmids and the cells were used for measuring cleaved
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caspase-3 by flow cytometry. Staurosporin was used as a positive control.
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