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Supplementary Information
Kaposi’s sarcoma herpesvirus lytic replication compromises
apoptotic response to p53 reactivation in virus-induced lymphomas
Grzegorz Sarek, Ph.D.1*, Li Ma, MSc.1*, Juulia Enbäck, MSc.2, Annika Järviluoma,
Ph.D.3, Pascale Moreau, Ph.D.4, Jürgen Haas, MD, Ph.D.5, Antoine Gessain, MD,
Ph.D.6, Päivi J. Koskinen, Ph.D.7, Pirjo Laakkonen, Ph.D.2,8, †, and Päivi M. Ojala,
Ph.D.1*,9, †
1
Research Programs Unit, Genome-Scale-Biology, Biomedicum Helsinki, Institute of
Biomedicine, University of Helsinki, Finland, 2Research Programs Unit, Molecular Cancer
Biology, Biomedicum Helsinki, Institute of Biomedicine, University of Helsinki, Finland,
3
Department of Virology, Haartman Institute, University of Helsinki, Finland, 4Clermont
Université, Université Blaise Pascal, SEESIB, Clermont-Ferrand, CNRS, Aubière, France,
5
Divison of Pathway Medicine, School of Biomedical Sciences, University of Edinburgh,
UK, and Max-von-Pettenkofer Institute, University of Munich, Germany,
Unité
6
d’Epidémiologie et Physiopathologie des Virus Oncogènes, Département de Virologie,
Bâtiment Lwoff, Institut Pasteur, 75015, Paris, France, 7Turku Centre for Biotechnology,
University of Turku and Åbo Akademi University, Turku, Finland, 8K. Albin Johansson
Foundation, 9Finnish Cancer Institute, Helsinki, Finland.
† Shared correspondence
* Current address: Institute of Biotechnology, University of Helsinki, Finland
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Supplementary Methods
Antibodies and protein analysis
The following primary antibodies were used: anti-p53 (DO-1 and FL-393), anti-p21
(C-19G), anti-MDM2 (SMP-14, and 2A10), and anti-Bax (N-20), anti-p65 (C-20),
anti-Sp1 (PEP-2), anti-CDK7 (C-4) all purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA); anti-MDM2 (IF-2) from Oncogene Sciences (Cambridge, MA);
anti-LANA (HHV8-ORF73), anti-vIL-6, and anti K8.1 from ABI (Columbia, MD);
anti-tubulin (5H1) from BD Bioscience; anti-GAPDH (14C10) and anti-cleaved
caspase-3 (#9661) were from Cell Signaling (Danvers, MA). ORF59 and rabbit
polyclonal LANA antibodies were generous gifts from Dr. Chandran (Rosalind
Franklin University of Medicine and Science, Chicago, IL). The RTA/ORF50
polyclonal antibody was kindly provided by Dr. Ganem (University of California, San
Francisco, CA). HRP-conjugated antibodies specific for rabbit, mouse, or rat
immunoglobulins were purchased from Chemicon International (Temecula, CA).
Alexa Fluor 594 goat anti-rabbit and goat anti-mouse antibodies were from Molecular
Probes (Eugene, OR). Subcellular fractionation to cytoplasmic and nuclear
compartments and Western blotting were carried out as described previously (1).
Signal intensities of Western blots bands were quantified with the ImageJ 1.45
software (National Institute of Health, Bethesda, MD).
Real-time quantitative PCR
Total RNA was prepared by using the RNAeasy kit according to manufacturer’s
instructions (Qiagen, Valencia, CA). RT-PCR was performed with TaqMan Reverse
Transcription Reagents kit (Roche Diagnostics, Indianapolis, IN) according to
manufacturer’s protocol. Real-time PCR conditions and set of primers for LANA,
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ORF50/RTA, ORF25, K8.1, GAPDH, and human beta-actin were as described
previously (2, 3). Primers for CA9 were as reported earlier (4).
Indirect immunofluorescence
Cells were suspended in phosphate-buffered saline (PBS), washed twice with PBS,
and fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Cells
were washed twice with PBS + 3% (FCS) and permeabilized with 0.5% NP-40
(Sigma, St. Louis, MO) for 5 minutes at room temperature. Cells were applied onto
glass slides and allowed to dry before storage at –20°C. Immunofluorescence labeling
was performed as described (5). The fluorochromes were visualized with a Zeiss
Axioplan 2 fluorescent microscope (Carl Zeiss, Oberkochen, Germany). Images were
acquired with a Zeiss Axiocam HRc, using Zeiss AxioVision (version 4.5 SP1) and
Adobe Photoshop software (version 7.0; Adobe, San Jose, CA).
TUNEL assay
Apoptotic cells were detected by an in situ cell death detection kit (TMR red;
Roche Applied Science; Mannheim, Germany), according to the manufacturer's
instructions. The assay measures DNA fragmentation by immunofluorescence using
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling)
method at the single cell level. Five hundred cells per sample were counted to
quantify the percentage of apoptotic cells.
IC50 determination
Analysis of Nutlin-3 IC50 change upon lytic replication in PEL cells was performed
using GraphPad Prism 5 software (Graphpad Software, La Jolla, CA). The nutlin-3
concentrations that led to 50% of cell death (IC50 values) were determined by
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regression analysis of displacement curves with variable slope to the dose–response
data.
Statistics
Statistical analyses were performed using two-tail unpaired Student’s t test. P values
less than 0.05 were considered significant.
Supplementary Figure Legends
Supplementary Figure S1. Nutlin-3-induced stabilization of p53 and p21 was
attenuated in the non-responder mice. Tumor cells from the ascites of non-responder
and responder mice shown in Figure 2a were subjected to SDS-PAGE followed by
Western blot analysis for the expression of p53 and p21. Protein expression was
quantified digitally by using the ImageJ software and normalized to Sp1 expression
used as a loading control.
Supplementary Figure S2. Viral lytic replication did not induce hyperactivation of
NF-B pathway in PEL cells. (a) BC-3/NF-B-luc and BC-3 cells were treated with
DMSO (Ctrl), 20ng/ml TPA, or 100 M etoposide (Eto). Cells were fractionated into
cytoplasmic (C) and nuclear (N) extracts that were resolved on SDS-PAGE and
analyzed by immunoblotting with anti-p65 antibodies. The distribution of a
cytoplasmic marker GAPDH and a nuclear marker CDK7 was used to control the
purity of the fractions. (b) p65 signal in the cytoplasmic and nuclear fractions was
quantified, and normalized to the signal of GAPDH and CDK7, respectively. The
graph shows the nuclear/cytoplasmic ratio (N/C) of the p65 signal.
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Supplementary Figure S3. TPA treatment did not affect p53 levels in the KSHVnegative cells. KSHV-negative IHE lymphoblastoid cells were pretreated with TPA
(20 ng/ml) or vehicle (DMSO) for 18 hours followed by incubation in the presence or
absence of Nutlin-3 (7 M) for additional six hours. Whole-cell extracts were
separated by SDS-PAGE followed by immunoblotting with antibodies against p53,
and MDM2. Tubulin served as a loading control.
Supplementary Figure S4. Nutlin-3-induced p53 stabilization and apoptosis were
compromised upon viral reactivation by inducible expression of RTA. (a) BCBL-1
TREx-RTA cells were pretreated with vehicle (Tet) or tetracycline (Tet+) for 18
hours followed by incubation in the presence or absence of Nutlin-3 (15 M) for
additional six hours. Cells were analyzed by indirect immunofluorescence and
double-labeled for expression of the early lytic marker ORF59 and p53. Nuclei were
counterstained with Hoechst; scale bar, 20 µm. Arrowheads point to ORF59-positive
cells with down-regulated p53. (b) BCBL-1 TREx-RTA cells were exposed to vehicle
(Tet) or tetracycline (Tet+) for 24 hours to induce ORF50/RTA expression. Cells
were then released from tetracycline after aspirating all media and quickly washing
with complete RPMI and incubated with 15 M Nutlin-3 for 72 hours. Whole-cell
extracts were separated by SDS-PAGE followed by immunoblotting with antibodies
against p53, MDM2, active caspase-3, and ORF50/RTA. Tubulin served as a loading
control. (c) Cells treated as in (b), were exposed to Nutlin-3 (15 M) for 72 or 96
hours and cell death was assessed by trypan blue exclusion. Data is representative of
two independent experiments  SD.
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Supplementary Figure S5. Hypoxia led to KSHV lytic reactivation in PEL cells. (a)
BC-3 and JSC-1 cells were incubated under normoxia (21% O2) or hypoxia (1% O2)
for 96 hours. Cells were analyzed by indirect immunofluorescence and stained with
the early lytic marker ORF59. Number of ORF59 positive cells was quantified.
Quantification represents an average of three independent experiments ± SD.
(b) JSC-1 cells were incubated under normoxia (21% O2) or hypoxia (1% O2) for 48
hours and then incubated in the absence (Nutlin-3-) or presence (Nutlin-3+) of 7 M
Nutlin-3 for six hours. Whole-cell extracts were resolved by SDS-PAGE and
followed by immunoblotting for vIL-6 and p53. Tubulin served as a loading control
Supplementary Figure S6. Inhibition of viral replication restored p53 levels in
reactivated PEL cells. JSC-1 cells kept at normoxia (21% O2) or hypoxia (1% O2) for
48 hours were simultaneously treated with ganciclovir (GCV), cidofovir (CDV), or
vehicle (Ctrl). Whole-cell extracts were resolved by SDS-PAGE and followed by
immunoblot analysis against p53. Tubulin served as a loading control.
Supplementary Figure S7. Efficiency of inhibition of lytic replication and toxicity
by DHPCC-9. BC-3 cells were pre-treated with the indicated concentrations of
DHPCC-9 for 4 hours and then incubated with TPA for an additional 48 hours. Cells
were analyzed by indirect immunofluorescence and stained with the early lytic marker
ORF59. Number of ORF59 positive cells was quantified as an indication of induction
of lytic replication (black squares, solid line). Toxicity was determined by trypan blue
exclusion in the non-induced (no TPA) cells treated with DHPCC-9 for 52 hours
(black circles, dotted line).
Supplementary Figure S8. Effect of proteasome inhibitor MG132 on lytic
replication and p53 protein levels in PEL cells. (a) BC-3 and JSC-1 cells were
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cultured in the presence (MG132+) or absence (MG132) of 1 mM MG132
proteasome inhibitor for 8 hours. Whole-cell extracts were subjected to SDS-PAGE
followed by Western blotting for ORF50/RTA (short and long exposures) and vIL6.
The first lane shows BCBL-1 TREx-RTA cells used as a positive control for lytic
protein expression. (b) JSC-1 cells were pre-incubated under normoxia (21% O2) or
hypoxia (1% O2) for 48 hours followed by culture for an additional eight hours in the
presence (+) or absence (-) of 1µM MG132. Cells were subjected to Nutlin-3 (+) or
vehicle control (-) for the last six hours. Cells were harvested and the whole cell
extracts were subjected to SDS-PAGE followed by immunoblotting for p53 (short and
long exposures), MDM2, and vIL-6. Tubulin served as a loading control.
Supplementary References
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