Evangelou et al
SUPPLEMENTARY INFORMATION
Table of Contents
page
Materials and Methods
2
Immunohistochemistry
2
Indirect Immunofluorescence
3
Protein extraction and immunoblotting (IB)
4
Chromatin immunoprecipitation (ChIP) assay
5
Flow cytometric analysis (FACS)
7
Isolation of nucleic acids
7
Allelic imbalance analysis of microsatellite loci D9S161
8
K-Ras mutational analysis
8
Bioinformatic and Statistical analysis
9
SUPPLEMENTARY TABLES
10
Suppl Table 1
10
Suppl Table 2
11
Suppl Table 3
12
SUPPLEMENTARY REFERENCES
14
SUPPLEMENTARY FIGURES AND LEGENDS
17
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Evangelou et al
Materials and Methods
Immunohistochemistry (IHC)
The following antibodies were used: anti-p14ARF (1:100) (ab49166, Abcam), antip14ARF (1:100) (4C6/4, Cell Signaling), anti-p14ARF (1:500) (rabbit, Ab4,
NeoMarkers), anti-phospho-histone H2A.X (1:1000) (Ser139, 05-636, Millipore), antiATM-pS1981 (1:100) (sc47739, Santa Cruz,), anti-ATM-p (1:100) (mouse
monoclonal),7 anti-Chk2-pT68 (IHC 1:100) (NB100-92502, Novus Biologicals), antip53 (1:100) (DO-7, sc47698, Santa Cruz,), anti-p53 (1:100) (DO-1, sc-126, Santa
Cruz), anti-p53 (1:400) (CM5p, Novocastra), anti-p21WAF1 (IHC 1:200) (F-5,
sc6246Santa Cruz,), anti-p16INK4A (IHC 1:100)
(F-12, sc1661, Santa Cruz), anti-
p19ARF (1:100) (M-60, sc22784, Santa Cruz).
Immunohistochemistry was performed using the UltraVision LP Detection System
(#TL-060-HD,
Thermo
Scientific,
Bioanalytica,
Greece)
according
to
the
manufacturer’s instructions. Thin paraffin sections (4 μm) were deparaffinised in
xylene and rehydrated in a graded series of ethanol-aqueous solutions. Antigen
retrieval was carried out in 10mM citrate buffer (pH 6.0) by heating the slides for 25
min in a microwave oven. Endogenous peroxidase activity was blocked by incubating
the sections in 3% hydrogen peroxide in TBS for 10 min. The primary antibodies were
incubated overnight. For p21WAF1, p16INK4A, p14ARF a Labeling Index (LI) was
established, based on the proportion of positive nuclei since staining intensity was
strong. Evaluation and controls for γΗ2ΑΧ, Chk2-pT68, ATM-p, ATM and p53 have
been previously described.7-9
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For immunohistochemistry of human urinary bladder tissues and tumours, the
tissue sections were deparaffinized and processed for sensitive immunoperoxidase
staining with the primary antibodies against the individual markers, as follows: The
primary antibodies were incubated overnight, followed by detection using the
Vectastain Elite kit (Vector Laboratories, Burlingame, CA, USA) and nickel sulphate
enhancement without nuclear counterstaining.7,9 Immunostaining patterns on each slide
were scored as reported previously,7,9 and the threshold for scoring as enhanced
expression was as indicated above.
Indirect Immunofluorescence (IF)
Indirect IF analysis was performed according to a published protocol.11 Briefly,
cells cultured on coverslips were fixed in 4% formaldehyde and permeabilized by 0.1%
Triton X-100 in two consecutive steps, each for 15 minutes at RT. After washing with
PBS, cells were blocked for 30 minutes in 10% foetal calf serum. The following
primary antibodies were used: mouse monoclonal antibody against histone H2A.X
phosphorylated at serine 139 (γH2AX; Abcam, ab22551, 1:500), mouse monoclonal
antibody against Cytochrome c (BD Pharmingen, 556432, 1:100), rabbit 53BP1 (Santa
Cruz, sc-22760, 1:1000), anti-E2F1 antibody (Santa Cruz, sc-251, 1:1000). Incubation
with primary antibodies was performed for 60 minutes at RT and then cells were
washed with PBS. Secondary antibodies Alexa Fluor® 488 goat anti-rabbit
(Invitrogen, #A11034, 1:500) and Alexa Fluor® 568 goat anti-mouse (Invitrogen,
#A110-31, 1:500) were then applied for 60 minutes at RT, followed by final wash in
PBS. Coverslips were mounted in Vectashield Mounting Medium with 100 ng/ml of
4,6-diamidino-2-phenylindole (DAPI)
(Vector
Laboratories,
#H-1200).
Image
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Evangelou et al
acquisition of multiple random fields was automated on a Scan^R screening station
(Olympus, Germany) and analyzed by using Scan^R (Olympus, Germany) analysis
software, or a Zeiss Axiolab fluorescence microscope equipped with a Zeiss Axiocam
MRm camera and Achroplan objectives while image acquisition was performed with
AxioVision software release 4.7.1.
Protein extraction and immunoblotting (IB)
The following antibodies were applied: anti-p14ARF (1:100) (Abcam), anti-ATM
(1:500) (Millipore), anti-γH2AX (1:500) (Millipore), anti-H2AX (1:500) (Millipore),
anti-Chk2-pT68 (1:100) (Novus Biologicals), anti-p53 (1:100) (Santa Cruz), antip21WAF1 (1:200) (Novocastra), anti-p16INK4A (IHC 1:100) (Santa Cruz), anti-p19ARF
(1:100) (Santa Cruz), anti-β-catenin (1:500) (Santa Cruz), anti-Ha-Ras (1:100) (Santa
Cruz), anti-β-actin (1:1000) (Cell Signaling Tech), anti-alpha-tubulin (1:10,000)
(Sigma), anti-CDK7 (1:1000) (Abcam).
Treated cells were lysed and protein lysates were subjected to IB analyses as
previously described.11 Briefly, cells were homogenized in 50 mmol/L HEPES, pH 7.5,
150 mmol/L NaCl, 15 mmol/L ß-mercaptoethanol, 0.5 mmol/L phenylmethanesulfonyl
fluoride (PMSF), 0.1% Nonidet P-40 (Sigma) and the solution was centrifuged at
12500 rpm (1000 x g) at 4°C for 10 minutes. The supernatant was isolated and adjusted
to 1μg/ml protease and phosphatase inhibitors (Pierce). The pellet was diluted in
10mM HEPES pH 7.9, 1.5mM MgCl2, 10mM KCl, 0.5mM DTT, and 1.5mM PMSF,
incubated in ice for 30 min and centrifuged at 12500 rpm (1000 x g) at 4°C for 10
minutes. Total protein concentration was estimated using the Bio-Rad Protein Assay
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(Bio-Rad). 30 μg of protein extracts were adjusted with Laemmli Buffer (Sigma) and
loaded on SDS poly-acrylamide/bis-acrylamide gels (PAGE).
Gel electrophoresis and transfer to PVDF membrane (Millipore) were carried out
according to standard methods. Blots were incubated for 1h in 5% non-fat milk in
TBS-0.1% (v/v) Tween-20 solution at room temperature. Primary antibody solution in
0.5% non-fat milk in TBS supplemented with 0.1 % (v/v) Tween-20 at room
temperature was applied on membranes overninght, followed by a 45 min incubation
with the appropriate alkaline phosphatase conjugated secondary antibody (1:5,000
dilution) (Promega) at room temperature. Signal visualization was carried out with
application
in
nitro
blue
tetrazolium/5-bromo-4-chloro-3-indolylphosphate
(NBT/BCIP) solution (Molecular Probes).
Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed as previously described.S71 3×106 normal HBECs
transiently transduced with the pBabe-Ha-RasV12 or transfected with anti-p53 siRNA,
3×106 immortalized (hTERT/Cdk4) HBECs and immortalized HBECs transfected with
K-RasV12, respectively, were crosslinked with 1% formaldehyde for 10 min at 37oC.
Crosslinking was stopped by addition of glycine to a final concentration of 125 mM for
5 min at 37oC. Cross-linked cells were washed twice in ice-cold PBS and scraped in 1
ml PBS containing protease and phosphatase inhibitors. Cells were collected by
centrifugation for 5 min, 2,000 rpm and the pellet was resuspended in 600 μl of buffer
(50 mM Tris-HCl pH 8.0, 85 mM KCl and 0.5% NP40) and incubated for 10 min on
ice. The lysate was centrifuged for 5 min at 5,000 rpm and the pellet resuspended in
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600 μl of lysis buffer (50 mM Hepes-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA pH
8.0, 1% Triton-X, 0.1% Sodium Deoxycholate and 0.1% SDS) containing protease and
phosphatase inhibitors. Lysates were sonicated to shear DNA to an average fragment
size of 500 – 1,000 bp. The debris was pelleted by 5 min centrifugation at 13,000 rpm
at 4oC and the soluble chromatin material was precleared with salmon sperm
DNA/50% protein A agarose slurry (Millipore). An anti-E2F1 antibody (sc-251 X,
Santa Cruz) was used for the immunoprecipitation. After overnight incubation the
immune complexes were harvested with 60 μl salmon sperm DNA/50% protein A
agarose slurry (Millipore) for 2 h at 4oC. The beads were washed sequentially for 5
min each at room temperature in 1 ml lysis buffer without SDS, in 1 ml lysis buffer
plus 500 mM NaCl, in 1 ml buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0,
250 mM LiCl, 1% NP40, 1% sodium deoxycholate) and finally twice in TE. Immune
complexes were eluted with 200 μl (2 times of 100 μl each) elution buffer (1% SDS,
50 mM Tris-HCl pH 7.5, 10 mM EDTA) and incubated for 5 min at 65 oC. The pooled
eluates were incubated with RNAse for 1 h and 4 h with proteinase K at 65 oC. DNA
was extracted by phenol-chlorophorm and precipitated with 10 μg glycogen and
ethanol overnight at -20oC. The DNA was resuspended in 10 μl TE. DNA from the
precipitated complexes was amplified by real-time PCR in a DNA-Engine-Opticon
(MJ-Research) cycler employing the Platinum SYBR Green qPCR SuperMix-UDG
(Invitrogen). A 310 bp fragment encompassing the human p14ARF promoter and a 5’distantly located (650bp) fragment of 210 bp representing a negative control (NC) for
E2F1 binding, were amplified with primers (and annealing temperatures) provided in
Suppl Table 3. As inputs we used products that corresponded to PCR reactions
containing 1% of the total chromatin extract used in the immunoprecipitation reactions.
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Flow cytometric analysis (FACS)
FACS analysis was conducted as previously described.11 HBECs were initially
infected with retroviruses containing pBabe-Ha-RasV12 and pBabe, supplemented with
10 μg/ml polybrene (Sigma), as previously described.11 After 24 hours cells were
transfected with control siRNA, anti-ATM siRNA, anti-ARF siRNA or anti-p53
siRNA, as previously described (Figure 3).11 After the next 2 days, cells from each
treatment were harvested with trypsinization and centrifuged at 1,000 rpm for 5 min at
room temperature. The pellet was resuspended in 500 μl PBS, fixed with 80% ethanol,
vortexed and stained with propidium iodide (50 μg/ml), in the presence of 5 mM
MgCl2 and 10 μg/ml RNAse A in 10 mM Tris–HCl pH 7.5. DNA content was assessed
on a FACS Calibur (Becton-Dickinson). Data from three independent measurements
were averaged and corresponding s.d. is also reported.
Isolation of nucleic acids
DNA extraction: cells were lysed in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5
mM EDTA, 1% SDS in the presence of proteinase K (0.1 mg/ml) until completely
dissolved. DNA was extracted with phenol/chloroform and RNase (Sigma) digestion,
followed by ethanol precipitation.11
RNA isolation with the Trizol reagent (Invitrogen) was employed following the
manufacturer’s instructions and has been previously described.11 Quality and quantity
of isolated nucleic acids was estimated by spectrophotometry, fluorometry and gel
electrophoresis.
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Allelic imbalance analysis of microsatellite loci D9S161
The D9S161 marker located within the chromosome locus 9p21-23, was examined
and LOH analysis was performed as previously described.S72 In brief, PCR reactions
were carried out using previous published primers and conditions.S72 PCR products
were electrophoretically separated on 10% polyacrylamide gels and stained with silver
nitrate. They were then analyzed by capillary chip electrophoresis on a 2100
Bioanalyser (Agilent). LOH was determined by densitometry, comparing the allelic
ratio of the normal against that of the cancerous tissue, as previously detailed
described.S72 A reduction of one of the alleles in the carcinomas was considered to
represent LOH. Alterations were confirmed by performing independent duplicate
reactions.
K-Ras mutational analysis
K-Ras mutation status at codons 12 and 13 was established by using PCR-RFLP
combined with Sanger sequencing. Briefly, duplicate semi-nested PCR reactions per
each sample were set up, as previously published.S73 Products from each duplicate,
were in turn employed in a RFLP reaction and a Sanger cycle sequencing reaction. For
assessment of K-Ras mutation status at codons 12 and 13 by RFLP, the semi-nested
amplified products were subjected to BstXI and XcmI digestion, correspondingly, as
previously described.S73 RFLP products were analyzed on 10% polyacrylamide gels
along with corresponding positive and negative codon 12 and 13 controls. Sanger
sequencing was performed on a 377 ABI PRISM sequencer following the
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manufacturer’s instructions. Cases that failed to give same results in the duplicates and
by both approaches were subjected to re-evaluation.
Bioinformatic and Statistical analysis
Data from SNP arrays were analyzed with the R package, Rseg.S74 For other
statistical analyses, ANOVA and Mann-Whitney tests were used by employing the
SPSS 17.0 software.
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SUPPLEMENTARY TABLES
Suppl. Table 1. Analysis of DDR markers (protein) and CDKN2A locus genes p14ARF
and p16INK4A (at both protein, n=110; and mRNA, n=40, levels) in human tissues from
urinary bladder tumours (Ta, T1 and T2-4 lesions) and normal urothelium (percentage
of cases with positive expression)* and frequency of genomic losses at CDKN2A locus
compared to the stage of tumour. Total number of tumours showing losses was 23
tumours derived from 14 patients. 22 Tumours from 17 patients showed no losses.
Marker
Normal
Ta
T1
T2-T4
p14ARF
0
0
12
27
p14ARF mRNA
0
4
11
63
pATM (DDR)
0
90
90
75
p16INK4A
0
60
31
70
p16INK4A mRNA
0
20
33
33
CDKN2A locus
deletion
0
50
56
50
.
* positive expression for protein markers indicates percentage of cases in which 10%
or more tumour cells scored positive; for mRNA levels positive cases are those with at
least two-fold higher expression compared to normal level. mRNA expression was
examined in only a subset (n=33) among the 103 lesions examined by
immunohistochemistry.
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Suppl. Table 2. Fold of change of p14ARF and p16INK4A mRNA expression levels
between normal bladder epithelium and cancerous samples as well as between Ta and
cancerous lesions
Normal vs cancer
Normal vs Ta
Ta vs cancer
p14ARF
fold change* p-value**
0.5
0.02
0.3
0.11
0.4
0.001
p16INK4A
fold change* p-value**
0.8
0.0006
0.7
0.006
0.04
0.3
*log2 fold change values
** t-test statistics
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Suppl. Table 3. Primers and annealing temperatures in the real-time RT-PCR analyses
Primer sequence
Annealing temp
Application
p14ARF:
(Fw1) 5΄-ATGGTGCGCAGGTTCTTGGTGA-3΄
(Rv1) 5΄-GGGGTCGGCGCAGTTGGGCTCA-3΄ 60°C
mRNA analysisS77
(Fw2) 5´-CCCTCGTGCTGATGCTACTGA-3´
(Rv2) 5´- CATGACCTGGTCTTCTAGGAAGC-3´ 60°C
mRNA analysisS78
(Fw3) 5´- CTACTGAGGAGCCAGCGTCTA-3´
(Rv3) 5´-CTGCCCATCATCATGACCT-3´
60°C
mRNA analysis
(Rv1) 5´-CACGGGTCGGGTGAGAGT-3´
60°C
mRNA analysis
(Fw2) 5’-CTGCCCAACGCACCGA-3´
(Rv2) 5’-CCATCATCATGACCTGGATCG-3´
60°C
mRNA analysisS78
(Fw3) 5´- GAGCAGCATGGAGCCTTC-3´
(Rv3) 5´-CTGCCCATCATCATGACCT-3´
60°C
mRNA analysis
60°C
mRNA analysis
50°C
mRNA analysis
58°C
mRNA analysis
p16INK4A:
(Fw1) 5´-CCAACGCACCGAATAGTTAC-3´
Noxa:
(Fw) 5’-GCTGGAAGTCGAGTGTGCTA-3’
(Rv) 5’-CCTGAGCAGAAGAGTTTGGA-3’
Puma:
(Fw) 5’-GACCTCAACGCACAGTA-3’
(Rv) 5’-CTAATTGGGCTCCATCT-3’
Bax:
(Fw) 5’-TGCTTCAGGGTTTCATCCAG-3’
(Rv) 5’-GGCGGCAATCATCCTCTG-3’
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PBGD:
(Fw) 5’- TGCAACGGCGGAAGAAAACA-3’
(Rv) 5’-GCAGATGGCTCCGATGGTG-3’
50-60°C
mRNA analysis
50-60°C
mRNA analysis
60°C
mRNA analysis
60°C
mRNA analysis
60°C
ChIP analysis
60°C
ChIP analysis
GAPDH:
(Fw) 5´-AGCCACATCGCTCAGACAC-3`
(Rv) 5´- GCCCAATACGACCAAATCC -3´
p21WAF1:
(Fw) 5´-TCACTGTCTTGTACCCTTGTGC-3´
(Rv) 5´- GGCGTTTGGAGTGGTAGAAA-3´
p53:
(Fw) 5´- CCCCAGCCAAAGAAGAAAC-3´
(Rv) 5´- AACATCTCGAAGCGCTCAC-3´
Promoter p14ARF:
(Fw) 5’-TGAGGGTGGGAAGATGGTG-3’
(Rv) 5’- CCCCTTAACTGCAGACTGG -3’
Negative control promoter p14ARF:
(Fw) 5’-GGACATGGAGGGGGAGACC-3’
(Rv) 5’- CTTCTTCCTCTTTCCTCTTCC -3’
.
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Suppl. Figure Legends
Suppl. Figure 1. a. The DNA Damage Response (DDR) is activated earlier than ARF
during murine pancreatic carcinogenesis from PanIN-1, whereas ARF is upregulated in
PanIN2-3. Insets depict areas of higher magnification. b. The DDR activation precedes
ARF expression in an Ncst-/- mouse skin model. Insets depict areas of higher
magnification Scale =50 μm.
Suppl. Figure 2. Representative immunohistochemical results of ARF expression,
assessed with two different anti-p14ARF antibodies in serial sections obtained from a set
of five human Head and Neck carcinoma cases comprising the whole spectrum of
precancerous and cancerous lesions, located in adjacent regions within the same
section and mounted on the same slide. The absence/low immunohistochemical
expression of ARF in normal, hyperplastic and dysplastic epithelium in adjacent
regions and its elevated immunolabeling in the carcinoma located near the
corresponding precancerous lesions were observed with both antibodies. This indicates
rather low ARF protein expression in the early stages rather than high working
thresholds of both ARF antibodies. Scale =50 μm.
Suppl. Figure 3. Affymetrix Exon Array 1.0 probeset distribution in CDKN2A locus.
CDKN2A p16INK4A and p14ARF transcripts were profiled in 11 normal urothelium
samples and 44 bladder tumours using the Affymetrix Exon 1.0 ST arrays. The probesets 3201480 and 3201481 are specific for estimating p14ARF and the probe-sets
3201464-67 are specific for estimating p16INK4A levels.
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Evangelou et al
Suppl. Figure 4. a. p16INK4A is frequently deleted from early stages of laryngeal
carcinogenesis. Decreased p16INK4A immunostaining, due to LOH at the CDKN2A
locus, from the stage of dysplasia in a representative case of head and neck squamous
cell carcinoma. Scale =50 μm. b. Loss of heterozygosity (red circle) was found in
61.5% of D9S171 informative cases (8/13).
Suppl. Figure 5. Analysis of DDR markers versus ARF expression in human
pancreatic carcinoma samples containing the whole spectrum of pancreatic
carcinogenesis. Positive p16INK4A immunostaining is exhibited in all lesions, confirmed
also by absence of LOH (D9S171). These cases harboured K-Ras mutations from
PanIN-1, the earliest preneoplastic stage. Intense γH2AX expression is observed from
PanIN-1, whereas ARF is robustly activated from PanIN-3. Scale =50 μm.
Suppl. Figure 6. a. Positive immunostaining for DDR and ARF in actinic keratosis
(n=5), with DDR activation being more intense than ARF. Bars represent the
percentage expression for each protein in normal tissue, actinic keratosis, in situ
carcinoma (Bowen disease) and tumour areas, respectively. b. DDR activation
precedes ARF upregulation during carcinogenesis. Growth factor [basic fibroblast
growth factor (bFGF), stem-cell factor (SCF) and endothelin] induced (FSE) reversible
hyperplasias from human skin xenografts in mice along with their controls were
analyzed immunohistochemically for γH2AX, Chk2-pT68 and p14ARF. NT=nontreated, Scale =50 μm.
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Suppl. Figure 7. Introduction of E2F1 in BJ cells induces senescence (% SA-b-gal
positive cells) that is effectively bypassed after treatment with the specific ATM
inhibitor Ku55933 (see Material and Methods sections).
Suppl. Figure 8. Schematic presentation of the time course followed for the employed
experimental procedures depicted in Figures 3, 5 and 8.
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