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Supplementary Information
Cells
Human Osteosarcoma U-2 OS cells and Saos-2 cells, human non-small-cell lung
carcinoma H1299 cells, MCF-7 breast adenocarcinoma cells and human foreskin
fibroblasts were maintained at 5% CO2 in Dulbecco's modified Eagle's medium
(Cambrex) supplemented with 10% fetal bovine serum (FBS) (Perbio), 1% penicillinstreptomycin (Cambrex), and 1% L-glutamine (Cambrex). MCF10A cells were cultured
in a 1:1 mix of Hams Nutrient Mixture F12 (Cambrex) and DMEM supplemented with
5% FBS, 10 g/ml insulin (Sigma), 20ng/ml Epidermal Growth Factor (Sigma), 5g/ml
hydrocortisone (Sigma) and penicillin/streptomycin/L-glutamine as above.
U-2 OS cells containing stably transfected p53 and p52 RNAi or control plasmids
were created as follows. U-2 OS cells were plated onto 3.5cm dishes to achieve 50%
confluency. The next day cells were transfected, with 1.5µg of pSilencer plasmids
(Ambion) expressing either p53 or control siRNAs, using GeneJuice transfection reagent
(Novagen) according to manufacturers instructions. 48 hours post transfection cells were
selected with hygromycin B. Cells were continously cultured in the presence of 200µg/ml
of hygromycin B to maintain selection. Cells used in Fig. 8 were from a pool of stable
clones. Data shown in Figure 10 is from a purified clone of cells and is repreentative of
data from two independently derived clones of p52 siRNA expressing cells.
H1299 cells containing stably transfected p53 (H1299w/tp53) have been described
previously (Rocha et al., 2003) and were grown in the presence of 400 µg/ml of
hygromycin B and 400 µg/ml of G418. Induction of p53 was achieved by treating cells
with 100 µM IPTG (Melford Laboratories) for the indicated times.
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Plasmids
p21 luciferase plasmids were obtained from David Gillespie (Beatson Cancer Institute,
Glasgow) and were originally from the Xiao-Fan Wang laboratory (Duke University).
DR5 luciferase plasmids were obtained from Toshiyuki Sakai (Kyoto Prefectural
University of Medicine) and Spencer Gibson (University of Manitoba). The p53 deletion
plasmids used for in vitro translation (Supp. Fig. 2B) were provided by Professor David
Lane (Dundee).
p52 RNAi, p53 RNAi and control plasmids used in transient transfection
experiments were generated using the pBS-U6 plasmid (Girdwood et al., 2003) digested
with BamH1 (5’) and HindIII (3’) with the appropriate oligo ligated in. p53 RNAi and
control plasmids used to generate stable cell lines were created using the pSilencer
plasmid (Ambion) using a 5’ BamH1 site and a 3’ HindIII site. RSV p52 and RSV p100
expression plasmids have been described previously (Rocha et al., 2003).
The GFP-p52, HA-p52 and HA-p52-DBM plasmids were created in pEGFP
(Living colours, Clontech) and pCMV5 HA respectively, by Adel Ibrahim and Mark
Peggie of the University of Dundee School of Life Sciences cloning service.
Antibodies
Antibodies used were: anti-p53 DO-1 and anti-HA tag (12CA5) (Cancer Research-UK),
anti-phospho-Ser-15 p53 (9284, New England Biolabs), anti-p21 (sc-397, Santa Cruz),
anti-p52 monoclonal and anti-p52 polyclonal (05-361 & 06-413, Upstate Biotechnology),
anti-p52 polyclonal (sc-848, Santa Cruz), anti-p50 NF-B (06-886, Upstate
Biotechnology), anti-RelA (sc-372, Santa Cruz), anti-Bcl-3 (sc-185, Santa Cruz), anti-actin (A5441, Sigma), anti-Mdm2/Hdm2 (OP46, Calbiochem), anti-Cyclin D1 (556470,
BD Pharmingen) and anti-GFP (1181446001, Roche). The anti-c-Rel and anti-RelB
antibodies were provided by Nancy Rice, National Cancer Institute, Frederick, Md, USA.
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Antibodies used in ChIP assays were anti-p52 polyclonal (sc-848, Santa Cruz), anti-p53
DO-1 (Cancer Research-UK), anti-Acetyl histone H3 (06-599, Upstate Biotechnology),
anti-Gal4 (sc-510, Santa Cruz), anti-p300 (554215, BD Pharmingen) and anti-HDAC1
(06-720, Upstate Biotechnology).
Caspase 3 assays
Caspase 3 assays were performed using the CASPACE kit from Promega and were
performed according to manufacturers instructions.
Flow cytometric analysis of cell cycle distribution.
Adherent and detached cells were harvested, pooled, washed once in phosphate-buffered
saline (PBS), and fixed in ice-cold 70% (vol/vol) ethanol in distilled water. Cells were
then washed twice in PBS (plus 1% (wt/vol) bovine serum albumin) and resuspended in
PBS containing 0.1% (vol/vol) Triton X-100, 50 µg of propidium iodide per ml and 50
µg of RNase A per ml. After incubation at room temperature for 20 min, cells were
analyzed for cell cycle distribution with a FACS Calibur flow cytometer (fluorescenceactivated cell sorter; FACS) and CellQuest software (Becton Dickinson). Red
fluorescence (585 ± 42 nm) was evaluated on a linear scale, and pulse width analysis was
used to exclude cell doublets and aggregates from the analysis. Cells with a DNA content
between 2N and 4N were designated as being in the G1, S, or G2/M phase of the cell
cycle. The number of cells in each compartment of the cell cycle was expressed as a
percentage of the total number of cells present.
Chromatin Immunoprecipitation (ChIP)
Cells were grown to 70% confluency and cross-linked with 1% formaldehyde at room
temperature for 10 min. Glycine was added to a final concentration of 0.125M for 5 mins
at room temperature. Cells were washed twice with 10 ml of ice-cold phosphate-buffered
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saline and then scraped into 2 mls ice cold harvest buffer (PBS, 1mM
phenylmethylsulfonyl fluoride (PMSF), 1 g/ml leupeptin, 1 g/ml aprotinin) before
being centrifuged at 1000rpm in an Avanti benchtop centrifuge at 4°C for 10 minutes.
The supernatant was removed and the pellet was resuspended in 0.5 ml of lysis buffer
(1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, 1 mM PMSF, 1 g/ml leupeptin, 1
g/ml aprotinin) and left on ice for 10 minutes. Samples were then sonicated at 4°C
seven times. Each sonication was for 20 seconds with a 1 minute gap between each
sonication. Supernatants were recovered by centrifugation at 12,000 rpm in an eppendorf
microfuge for 10 min at 4°C before being diluted 10 fold in dilution buffer (1% Triton X100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1). Samples were then precleared for 2 hours at 4°C with 2 g of sheared salmon sperm DNA and 20 l of protein
A-Sepharose (50% slurry). At this stage, 10% of the material was kept and stored at –
20°C as Input material. Immunoprecipitations were performed overnight with specific
antibodies (3g), with the addition of BRIJ-35 detergent to a final concentration of 0.1%.
The immune complexes were captured by incubation with 30 l of protein A-Sepharose
(50% slurry) and 2 g salmon sperm DNA for 1 hour at 4°C. The immunoprecipitates
were washed sequentially for 5 minutes each at 4°C in Wash Buffer 1 (0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), Wash Buffer 2
(0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl),
and Wash Buffer 3 (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10
mM Tris-HCl, pH 8.1). Beads were washed twice with Tris borate-EDTA (TBE) buffer
and eluted with 100 l of Elution Buffer (1% SDS, 0.1 M NaHCO3). For Re-ChIP
experiments 25µl of ReChIP buffer (Dilution Buffer, 10mM DTT) was added to beads
following washes and incubated at 37°C for 30 minutes. The sample was then diluted 40
times in dilution buffer and immunoprecipitations, washes and elution were performed as
before.
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To reverse the crosslinks, samples, including 'Input', were incubated in 0.2M
NaCl at 65 °C overnight. Supernatants were then incubated for 1 hour at 45°C with
Proteinase K (20 g each), 40mM Tris-HCl pH6.5, and 10mM EDTA. DNA was
cleaned up using PCR Purification columns (Qiagen) according to manufacturers
instructions. 5l of DNA was used from a 50µl DNA preparation and subjected to 35
cycles of PCR amplifications (apart from Figure 7 where 10l was used). Control
regions for each gene were subjected to 40 cycles of PCR Amplification.
For the experiments in Figure 9, U-2OS cells on a 15cm dish were transfected
using Genejuice with 10g of HA, HA-p52 or HA-p52-DBM plasmids. After 48 hours,
cells were harvested and processed as above for ChIP analysis.
Primers and oligonucleotides
Semi-Quantitative PCR
Cyclin D1 sense CCATTCCCTTGACTGCCCGAG
Cyclin D1 antisense GACCAGCCTCTTCCTCCAC
Chk1 sense CCTTTGTGGAAGACTGGGACTTGG
Chk1 antisense CATCTTGTTCAACAAACGCTGACG
DR5 sense GCGCCCACAAAATACACCGACGAT
DR5 antisense GCAGCGCAAGCAGAAAAGGAG
Gadd45 sense TGCTCAGCAAAGCCCTGAGT
Gadd45 antisense GCAGGCACAACACCACGTTA
p21 sense CCTGGGACCTCACCTGCTCTGCTG
p21 antisense GCAGAAGATGTAGAGCGGGCCTTT
p53 sense CCCAAGCAATGGATGATTTGA
p53 antisense GGCATTCTGGGAGCTTCATCT
Puma sense CAGACTGTGAATCCTGTGCT
Puma antisense ACAGTATCTTACAGGCTGGG
Bcl3 sense TCAAGAACTGCCACAACGACAC
Bcl3 antisense GCAGATCTTGGACTCATGAGG
NFKB1 sense TCCCATGGTGGACTACCTGG
NFKB1 antisense ATAGGCAAGGTCAAGGTGC
NFKB2 sense AAGGACATGACTGCCCAATTTAAC
NFKB2 antisense ATCATGGATGGGCTGGGAGG
RelA sense CTCGGTGGGGATGAGATCTTC
RelA antisense CCGGTGACGATCGTCTGTATC
RelB sense CATCGAGCTCCGGGATTGT
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RelB antisense CTTCAGGGACCCAGCGTTGTA
c-rel sense AGAGGGGAATGCGTTTTAGATACA
c-rel antisense CAGGGAGAAAAACTTGAAAACACA
GAPDH sense GGTCGTATTGGGCGCCTGGTCACC
GAPDH antisense CACACCCATGACGAACATGGGGGC
ChIP primers
CyclinD1 sense TCCCATTCTCTGCCGGGCTTTGATC
CyclinD1 antisense GCTGGTGTTCCATGGCTGGGGC
p21 sense GTGGCTCTGATTGGCTTTCTGGCC
p21 antisense CAGCCCTGTCGCAAGGATCCTGCTGG
p21 control sense GATGGCTATGTCGGTGAAGCTC
p21 control antisense CCTTTGGCCACACTGAGGAATG
Chk1 sense AAGCTCCAACATAAACTGCTCGCTTTC
Chk1 antisense GTGCTTTGTAAACCTCAGAGTGGGGTACT
Chk1 control sense GGGGAGTTCCGTGTGGAC
Chk1 control antisense ACTGGCTTCTGTGTAAACCTGTCC
DR5 sense GATCTACTTTAAGGGCTGAAACCCACGG
DR5 antisense GGCGACAACGAGCACAAGGGTCTTGGG
DR5 control sense TCCGAACACATCCGAACATCAG
DR5 control antisense TCCCTGCACCCTTGCTACACTTAG
Gadd45 sense GGATCTGTGGTAGGTGAGGGTCAGG
Gadd45 antisense GGAATTAGTCACGGGAGGCAGTGCAG
Gadd45 control sense GGAGTTGGAGTTGTCAGGAAAAAGGG
Gadd45 control antisense GGTTGTGGTCTTTCAGGCCTCCACACC
Puma sense GGAGGAAAGCTGAGGAGTTCCCAATGTTGC
Puma antisense CTTACTGGGTCTCACCCAATCGCAATCGCC
Puma control sense GTGTAAGTGTGAGCCCCATCAG
Puma control antisense ACCCCCAGCGATGCGTAC
GAPDH sense CGGTGCGTGCCCAGTTG
GAPDH antisense GCGACGCAAAAGAAGATG
siRNA sequences (upper strand only is shown)
Control CAGUCGCGUUUGCGACUGG
p52/p100_A (129) CAGCCUAAGCAGAGAGGCU
p52/p100_B (249) CUACGAGGGACCAGCCAAG
p52/p100_C (887) GAUGAAGAUUGAGCGGCCU
p50/p105 GGGGCUAUAAUCCUGGACU
RelB UUGGAGAUCAUCGACGAGU
Bcl-3 CAACCUACGGCAGACACCG
p53 GACUCCAGUGGUAAUCUAC
Cyclin D1 UGUGUGCAGAAGGAGGUCTT
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Oligonucleotides used for siRNA plasmids – sequences in italics and underlined are
RNAi sequence
Control sense
GATCCCCAACAGTCGCGTTTGCGACTGGTTCAAGAGACCAGTCGCAAACGCGAC
TGTTTTTTTGGAAA
Control antisense
AGCTTTTCCAAAAAAACAGTCGCGTTTGCGACTGGTCTCTTGAACCAGTCGCAA
ACGCGACTGTTGGG
p52/p100_1 sense
GATCCCCAACAGCCTAAGCAGAGAGGCTTTCAAGAGAAGCCTCTCTGCTTAGGC
TGTTTTTTTGGAAA
p52/p100_1 antisense
AGCTTTTCCAAAAAAACAGCCTAAGCAGAGAGGCTTCTCTTGAAAGCCTCTCTG
CTTAGGCTGTTGGG
p52/p100_2 sense
GATCCCCAACTACGAGGGACCAGCCAAGTTCAAGAGACTTGGCTGGTCCCTCGT
AGTTTTTTTGGAAA
p52/p100_2 antisense
AGCTTTTCCAAAAAAACTACGAGGGACCAGCCAAGTCTCTTGAACTTGGCTGGT
CCCTCGTAGTTGGG
p52/p100_3 sense
GATCCCCAAGATGAAGATTGAGCGGCCTTTCAAGAGAAGGCCGCTCAATCTTCAT
CTTTTTTTGGAAA
p52/p100_3 antisense
AGCTTTTCCAAAAAAAGATGAAGATTGAGCGGCCTTCTCTTGAAAGGCCGCTCA
ATCTTCATCTTGGG
p53 sense
GATCCCCAAGACTCCAGTGGTAATCTACTTCAAGAGAGTAGATTACCACTGGAGT
CTTTTTTTGGAAA
p53 antisense
AGCTTTTCCAAAAAAAGACTCCAGTGGTAATCTACTCTCTTGAAGTAGATTACCA
CTGGAGTCTGGG
References
Girdwood, D., Bumpass, D., Vaughan, O. A., Thain, A., Anderson, L. A., Snowden, A.
W., Garcia-Wilson, E., Perkins, N. D., and Hay, R. T. (2003). p300 transcriptional
repression is mediated by SUMO modification. Mol Cell 11: 1043-1054.
Rocha, S., Martin, A. M., Meek, D. W., and Perkins, N. D. (2003). p53 Represses cyclin
D1 transcription through down regulation of Bcl-3 and inducing increased association of
the p52 NF-B subunit with histone deacetylase 1. Mol Cell Biol 23: 4713-4727.
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