Journal of Biomolecular Screening
The online version of this article can be found at:
2011 16: 251 originally published online 13 January 2011J Biomol Screen
Kenneth W. Yip, Michael Cuddy, Clemencia Pinilla, Marc Giulanotti, Susanne Heynen-Genel, Shu-Ichi Matsuzawa and John C.
A High-Content Screening (HCS) Assay for the Identification of Chemical Inducers of PML Oncogenic Domains
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© 2011 Society for Laboratory Automation and Screening www.slas.org 251
(pml-nBs), kremer bodies, nd10 (nuclear domain 10), or
nuclear dots (nds), are ~0.2- to 1-µm subnuclear structures
present in a wide variety of cell types.1,2 pml is required for the
formation of pods, and more than 30 proteins either transiently or
constitutively co-localize with pml in pods.2 the significance of
the PML gene was first noted in acute promyelocytic leukemia
(apl), wherein the vast majority of cases are characterized
romyelocytic leukemia protein (pml) oncogenic
domains (pods), also known as pml nuclear bodies
by t(15;17) chromosomal translocations that result in a pml-
rarα fusion protein that disrupts pml function by delocalizing
pml into microspeckled nuclear structures (the reciprocal
rarα-pml fusion protein disrupts rarα function).3 expression
of pml-rarα in the promyelocytic/myeloid compartment of
transgenic mice causes leukemia with apl features, underscor-
ing the tumor-suppressive activity of pods.1-3
pml plays an essential role in both caspase-dependent and
caspase-independent cell death.2 pml–/– mice are resistant to
apoptosis induced by numerous stimuli and have an increased
tumor incidence.4,5 pml contributes to cell death induced by
γ-radiation, the primary treatment modality for a wide variety
of tumors.6 interferons and arsenicals (e.g., as2o3) increase the
number and size of pods per cell and sensitize and/or induce
apoptosis in a variety of tumor cell types.7,8 interestingly, pml
confers direct resistance to many viruses, and numerous viruses
have evolved mechanisms for disrupting pod formation.9
among the proteins that localize to pods are p53, daxx,
and recQ dna helicase. the tumor suppressor p53 requires
acetylation by cBp/p300 at pods for the induction of apopto-
sis.2,10 daxx localization to pods increases pten nuclear
localization and pten tumor suppressor activity by inhibiting
1sanford-Burnham medical research institute, la Jolla, ca, usa.
2torrey pines institute for molecular studies, san diego, ca, usa.
3torrey pines institute for molecular studies, port saint-lucie, Fl, usa.
4conrad prebys center for chemical genomics, sanford-Burnham medical
research institute, la Jolla, ca, usa.
*current address: university of toronto, toronto, canada.
received aug 31, 2010, and in revised form oct 19, 2010. accepted for publi-
cation oct 21, 2010.
Journal of Biomolecular screening 16(2); 2011
A High-content Screening (HcS) Assay for the
Identification of chemical Inducers of PML
oncogenic domains (Pods)
KennetH W. YIP,1,* MIcHAeL cuddY,1 cLeMencIA PInILLA,2 MArc GIuLAnottI,3
SuSAnne HeYnen-GeneL,4 SHu-IcHI MAtSuzAWA,1 and JoHn c. reed1,4
pml is a multi-functional protein with roles in tumor suppression and host defense against viruses. When active, pml local-
izes to subnuclear structures named pml oncogenic domains (pods) or pml nuclear bodies (pml-nBs), whereas inactive
pml is located diffusely throughout the nucleus of cells. the objective of the current study was to develop a high content
screening (Hcs) assay for the identification of chemical activators of pml. We describe methods for automated analysis of
pod formation using high throughput microscopy (Htm) to localize pml immunofluorescence in conjunction with image
analysis software for pod quantification. using this Hcs assay in 384 well format, we performed pilot screens of a small
synthetic chemical library and mixture-based combinatorial libraries, demonstrating the robust performance of the assay.
Hcs counter-screening assays were also developed for hit characterization, based on immunofluorescence analyses of the
subcellular location of phosphorylated H2aX or phosphorylated cHk1, which increase in a punctate nuclear pattern in
response to dna damage. thus, the Hcs assay devised here represents a high throughput screen that can be utilized to
discover pod-inducing compounds that may restore the tumor suppressor activity of pml in cancers or possibly promote
anti-viral states. (Journal of Biomolecular Screening 2011;16:251-258)
Key words: pml, pod, nuclear bodies, apoptosis, high content screening
Yip et al.
252 www.slas.org Journal of Biomolecular Screening 16(2); 2011
Hausp-mediated pten deubiquitinylation.11 pten nuclear
exclusion is associated with cancer progression, and Hausp
overexpression coincides with pten nuclear exclusion in pros-
tate cancer.11 daxx also represses the transcription of various
antiapoptotic rel-B-associated genes, including ciap2, cFlip,
and Bfl-1 (a1), via histone deacetylase (Hdac) and dna meth-
yltransferase binding and recruitment.12,13 pods are required for
the maintenance of genomic stability in combination with pro-
teins such as recQ dna.14 notably, pml also suppresses the
anchorage-independent growth of transformed cells, is required
for hypophosphorylated rb-mediated cell cycle arrest, and
inhibits neoangiogenesis in human and mouse tumors.1,15
the fundamental roles of pods and pod-related proteins in
tumor suppression validate targeting pods for drug discovery.
pod formation is essential in interferon and arsenical cancer
therapy, especially for leukemias and multiple myeloma.2,16,17
interferons and arsenicals, however, induce a plethora of toxic
effects, limiting their effectiveness.17,18 the objective of the
current study was to develop a high-content screening (Hcs)
assay for the high-throughput identification of chemical activa-
tors of pods.
MAterIALS And MetHodS
Hela cells were originally obtained from atcc (manassas,
Va) and cultured in dulbecco’s modified eagle’s medium
(dmem; invitrogen, carlsbad, ca), with 10% fetal bovine
serum (FBs; clontech, mountain View, ca) and penicillin-
streptomycin (diluted according to the manufacturer’s specifica-
tions; invitrogen) at 37 °c, 5% co2. ppc-1 cells were cultured
similarly but with rpmi 1640 (invitrogen) instead of dmem.
the lopac1280 collection of 1280 pharmacologically active
single compounds was obtained from sigma-aldrich (st.
louis, mo). the torrey pines institute for molecular studies
combinatorial libraries are mixture-based libraries in posi-
tional scanning format, and they were dissolved in dimethylfor-
in total, 3150 cells/well (50-µl/well volume) were seeded
onto 384-well clear-bottom plates (greiner Bio-one, monroe,
nc) using the matrix Wellmate liquid dispenser (thermo Fisher
scientific, Hudson, nH) and incubated at 37 °c (5% co2). after
24 h, the Biomek FX laboratory automation Workstation
(Beckman coulter, Fullerton, ca) was used to add interferon-γ
(iFn-γ; 4 u/µl with either 0.1% dmso or dmF; r&d systems,
minneapolis, mn), dmso (0.1% final concentration; sigma-
aldrich), dmF (0.1% final concentration; sigma-aldrich), or
compounds. after 12 h, cells were washed with phosphate-
buffered saline (pBs), fixed with 4% formaldehyde (sigma-
aldrich) for 15 min, washed, permeabilized with 0.5% triton
X-100 (sigma-aldrich) for 5 min, washed, incubated with the
primary antibody diluted to 0.5 µg/ml in 5% bovine serum albu-
min (Bsa; thermo Fisher scientific) for 1 h, washed, incubated
with the secondary antibody diluted to 5 µg/ml in 5% Bsa for
1 h, washed, and placed in a 100-ng/ml dapi-pBs solution
(invitrogen) overnight. each wash step was a multiple fluid
change using the titertek map-c ii microplate Washer and
stacker (aspiration to 10 µl, 50 µl pBs addition, repeated a total
of 3 times; titertek, Huntsville, al).
For pml immunostaining, mouse monoclonal antihuman
pml primary antibody (pg-m3, santa cruz Biotechnology,
santa cruz, ca) was used with an alexa Fluor 488 chicken
antimouse igg secondary antibody (invitrogen). For phospho-
H2aX (ser139; p-H2aX or γ-H2aX) or phospho-chk1
(ser317) immunostaining, rabbit polyclonal antihuman phos-
phohistone H2a.X (ser139; diluted 1:400; cell signaling
technology, danvers, ma) or phospho-chk1 (ser317; diluted
1:400; cell signaling technology) primary antibodies were
used, respectively, with an alexa Fluor 568 goat antirabbit igg
secondary antibody (invitrogen).
plates were imaged using the Beckman coulter cell lab
ic-100 image cytometer with a 40× 0.6na elWd plan Fluor
dry (air) objective (6 images/well). the images (>200 cells/
well) were analyzed using the pod detection algorithm, which
was developed based on cytoshop (Beckman coulter) and
matlaB (mathWorks, natick, ma) software.
Hcs was performed at the conrad prebys center for
chemical genomics at the sanford-Burnham medical research
institute (la Jolla, ca).
Hcs performance was characterized using the following
equation: Z′ factor = 1 – (3σpositive + 3σnegative)/│(µpositive –
µnegative)│, where σpositive is the standard deviation of the positive
control, σnegative is the standard deviation of the negative control,
µpositive is the mean of the positive control, and µnegative is the
mean of the negative control.22
Development and optimization of the
PML-POD localization assay
immunostaining conditions were optimized for detection of
pml using a commercially available mouse monoclonal anti-
body. Hela cells were seeded in 384-well plates, treated with
either dmso or iFn-γ for 24 h, and immunofluorescently
HcS Assay for Pod Activators
Journal of Biomolecular Screening 16(2); 2011 www.slas.org 253
stained for pml to confirm iFn-γ-induced pml localization
into pods (Fig. 1A). iFn-γ-induced localization of pml into
pods is accompanied by the localization of various other pro-
teins to pods, such as daxx.23 thus, simultaneous immun-
ofluorescence detection of both pml and daxx in Hela cells
confirmed co-localization/formation of pods and validated
iFn-γ as a positive control (Fig. 1B). iFn-γ-induced pml and
daxx co-localization to pods was also confirmed to occur in
ppc-1 cells (observed by immunofluorescence; data not
to quantify the extent of pod formation (i.e., the number of
pods per cell, the intensity of pml localization, and the frac-
tion of cells per well with extensive numbers of pods) in an
automated fashion, the “pod detection algorithm” was devel-
oped using Beckman coulter cytoshop and mathWorks
matlaB software (Fig. 2A). First, the nuclear image (dapi
stain) was used to produce a “nuclear mask,” which identified
all nuclei in an image. this nuclear mask was applied to the
pml (“green”) image, and all green pixels outside of this
nuclear mask were eliminated. next, pods were outlined based
on the identification of green pixels with higher intensities than
their surrounding pixels (using cytoshop’s “aggregate
detection”), with the minimum size of a pod defined based on
iFn-γ control wells (cytoshop’s “object scale”). this number of
detected pods was then reported on a per nucleus basis and used
to determine the percentage of nuclei per image that were “pod
positive.” the value for the number of detected pods above
which a nucleus is considered pod positive was determined to be
4.0 by iteratively setting increasing threshold values and deter-
mining the Z′ factor for each threshold (on control plates).
to both further validate the algorithm, Hela cells were
treated with increasing concentrations of iFn-γ, stained with
anti-pml antibody, and imaged, and the percentage of pod-
positive cells was determined (Fig. 2B). using this automated
method, iFn-γ was determined to induce concentration-
dependent pod formation in Hela cells. manual counting of
pod-positive cells confirmed the algorithm-defined quantifi-
cation (data not shown). examples of whole-field images from
single wells (multiple images from single wells) are shown in
supplemental Figure s1 (online at http://jbx.sagepub.com/
to characterize the reproducibility of the assay, multiple
replicates were prepared (n > 80 per condition), where cells
were seeded into a 384-well plate using the matrix Wellmate
bulk liquid dispenser (3150 cells/well); treated with iFn-γ
(4 u/µl), dmso (0.1%), or nothing for 12 h; and immunos-
tained for pml, imaged, and analyzed. the Z′ factor was deter-
mined to be 0.64 or 0.65 using dmso or untreated cells,
respectively, as the negative control (Fig. 2c).
the Hcs assay was used to screen the lopac1280 library of
pharmacologically active compounds for pml activators (Fig.
3A). compounds that increased the number of pod-positive
nuclei by at least 50% (relative to the controls), as measured by
the pod detection algorithm, were considered hits. no hits
were identified when the library was screened at 5 µm.
moreover, dose-response curves (serial dilutions performed
from 250 µm to 0.25 µm) were generated for the 5 lopac1280
compounds inducing the most pod positivity, and the highest
value observed was less than 40% pod-positive nuclei (data
not shown). thus, the Hcs assay is not promiscuous with
respect to hit rate.
the tpims mixture-based combinatorial libraries were for-
matted and plated as a “scaffold ranking library,” which was
subsequently screened to determine the most active chemical
scaffolds before further screening/deconvolution of mixtures.24
the scaffold ranking library was formatted as 38 mixtures (dis-
solved in dmF), grouped according to scaffold, and repre-
sented 5,287,896 compounds (and several million peptides).
each mixture was present in 2 different wells at 2 different
concentrations. use of mixtures makes it readily feasible to
FIG. 1. pml and daxx localize to pml oncogenic domains (pods)
after interferon-γ (iFn-γ) treatment. (A) pml localizes to nuclear
bodies. Hela cells were seeded in 384-well plates (3150 cells/well),
treated (12 h) with dmso (0.1%) or iFn-γ (4 u/µl), immunostained
with mouse monoclonal antihuman pml and alexa Fluor 488 chicken
antimouse antibodies (green), and incubated in dapi (nuclear stain;
100 ng/ml; blue). cells were imaged using the cell lab ic-100 image
cytometer (40× 0.6na elWd plan Fluor objective). (B) pml and
daxx co-localize to pods. cells were treated and imaged as in A but
also immunostained for daxx (rabbit polyclonal antihuman daxx
primary antibody, alexa Fluor 568 goat antirabbit secondary antibody;
Yip et al.
254 www.slas.org Journal of Biomolecular Screening 16(2); 2011
screen large collections of compounds. this library was
screened at 5 µg/ml (~10 µm for average molecular weight
[mW] ≈500 g/mol) and 10 µg/ml (~20 µm for average mW
≈500 g/mol; Fig. 3B). the most active mixture was “1422,”
which induced >40% pod-positive cells.
the 1422 library contains compounds with an n-methyl
triamine scaffold in which chemical diversity was created at 3
positions, r1, r2, and r3. analysis of a collection of n-methyl
triamines, at which one of these diversity positions was fixed,
allowing the others to vary as a mixture of all possibilities built
FIG. 2. High-content screen (Hcs) development and optimization. (A) algorithm for detecting and quantifying pml oncogenic domains
(pods). dapi (a.1; blue), pml (a.2; green), and merged (a.3) images of interferon-γ (iFn-γ)–treated (4 u/µl; 12 h) Hela cells are shown.
the nuclear (dapi) image was used to produce a nuclear mask (B; blue). the nuclear mask (c; blue outline) was applied to the pml image.
green pixels outside of the nuclear mask were eliminated (d). Beckman coulter cytoshop software was used to estimate cellular area (e; red
outline) based on the nuclei. pod outlines (F; red) were identified based on differences in green pixel brightness. the number of detected pods
(g; red) was reported on a per nucleus basis. the percentage of pod-positive nuclei (>4.0 pods per cell) was reported. (B) Quantification of
iFn-γ-induced pod formation. Hela cells were seeded in a 384-well plate (3150 cells/well) and treated with increasing concentrations of iFn-
γ (12 h) as shown. cells were immunostained for pods (mouse monoclonal antihuman pml and alexa Fluor 488 chicken antimouse antibodies),
incubated in dapi (nuclear stain; 100 ng/ml), imaged using the Beckman coulter cell lab ic-100 image cytometer (40× 0.6na elWd plan
Fluor objective), and quantified using the described computerized algorithm. mean ± standard deviation are shown (n = 4 wells/data point, >200
cells imaged and quantified per well). (c) pod Hcs reproducibility assessment. Hela cells were cultured overnight in a 384-well plate (3150
cells/well seeded using the matrix Wellmate bulk liquid dispenser; treated with iFn-γ (4 u/µl), dmso (0.1%), or nothing for 12 h; and immu-
nostained for pml, imaged, and analyzed (n > 85 wells/condition). the Z′ factor is 0.64 or 0.65 using dmso or untreated cells, respectively, as
the negative control.
HcS Assay for Pod Activators
Journal of Biomolecular Screening 16(2); 2011 www.slas.org 255
FIG. 3. High-content screening (Hcs) for chemical activators of pml oncogenic domains (pods). (A) sample plate from the lopac1280 pod
localization screen. Hela cells were seeded in 384-well plates (3150 cells/well) and incubated at 37 °c overnight. the cells were then treated for
12 h with iFn-γ (4 u/µl; positive control), dmso (0.1%; negative control), or lopac1280 compounds (5 µm). plates were immunostained for
pml, imaged, and analyzed. the Z′ factor was 0.55. (the dotted line represents the mean value between the positive and negative controls.) (B)
tpims combinatorial library pod localization screen. cells were screened as described previously but with mixtures from the tpims scaffold
ranking library (5 µg/ml or 10 µg/ml, representing ˜10 µm or ˜20 µm, respectively, for molecular weight [mW] ≈500 g/mol). interferon-γ (iFn-
γ; 4 u/µl) was used as the positive control, and dimethylformamide (dmF; 0.1%) was used as the negative control. the Z′ factor was 0.6. (c-e)
combinatorial library deconvolution reveals the structure-activity relationship of n-methyl triamine compounds. deconvolution of the mixture-
based n-methyl triamine library was accomplished by the positional scanning method,23-26 where one of the substituents used to create the library
was fixed at positions (c) r1, (d) r2, or (e) r3, and the other positions were allowed to vary as mixtures of all possible substituents employed
in library construction. compounds were tested at 4 µg/ml (representing ˜8 µm for mW ≈500 g/mol). iFn-γ (4 u/µl) was used as the positive
control, and dmF (0.1%) was used as the negative control (striped bar). the Z′ factor was ~0.5.
Yip et al.
256 www.slas.org Journal of Biomolecular Screening 16(2); 2011
into the combinational library, revealed substituents at r1, r2,
or r3 that induced >50% pod-positive cells when tested at
4 µg/ml (~8 µm; Fig. 3c-e). successful implementation of
the Hcs assay for pod inducers for structure-activity relation
(sar) analysis of the n-methyl triamine combinatorial library
demonstrates the robust performance of the assay.
to complement the Hcs assay for pod activation, we also
devised 2 Hcs counterscreen assays using immunostaining for
the dna damage/repair–related proteins, H2aX and chk1.
H2aX is phosphorylated primarily by atm, which “senses”
double-stranded dna breaks, whereas chk1 is phosphorylated
primarily by atr, which “senses” single-stranded dna breaks
(a common intermediate found at sites of dna damage detec-
tion and repair pathways).25 immunofluorescence staining for
phospho-H2aX (ser139) (γ-H2aX) and phospho-chk1
(ser317), which are visible as nuclear aggregates, was used as
a counterscreen to test the specificity of the most active 1422
compounds. the similarity in antigen redistribution for H2aX
and chk1 compared to pml provides the basis for counter-
screens that eliminate false-positive compounds that might
affect pml or alter immunostaining patterns in a nonspecific
manner. in addition, because some types of dna-damaging
agents can stimulate pod formation,25-29 the phospho-H2aX
and phospho-chk1 immunofluorescence counterscreens serve
to eliminate compounds that operate as dna-damaging agents
and thus eliminate these from further consideration.
For these counterscreen Hcs assays, cytoshop algorithms
were used to quantify the intensity of nuclear phospho-H2aX
(ser139) (γ-H2aX) or phospho-chk1 (ser317) immunofluo-
rescence. as shown in Figure 4 (and Supplemental Figures
S2-S4 online at http://jbx.sagepub.com/content/by/supple-
mental-data), iFn-γ (pod-positivity control) induced pml
localization to pods without affecting the markers of dna
damage. similarly, 3 active n-methyl triamine (1422) com-
pounds synthesized based on results from the combinatorial
library analysis (1422-9, 1422-10, and 1422-62) induced pml
localization into nuclear aggregates without altering phospho-
H2aX or phospho-chk1, thus confirming the selectivity of
their pod-inducing activity. in contrast, cisplatin (dna dam-
age control) induced H2aX and chk1 phosphorylation without
affecting pod formation, whereas etoposide (dna damage
control) simultaneously induced pod formation, H2aX phos-
phorylation, and chk1 phosphorylation. staurosporine (a
broad-spectrum kinase inhibitor), dmso (solvent control), and
dmF (solvent control) had little effect on pod activation,
H2aX phosphorylation, or chk1 phosphorylation. altogether,
these results validate both the primary Hcs assay for detection
of pod activators and also the counterscreening assays for
elimination of dna damage-inducing compounds.
the current study describes an Hcs assay for compounds
that induce pml to localize to pods, subnuclear structures
involved in a variety of tumor-suppressive pathways, and host
defense against some types of viruses. after developing algo-
rithms for automated analyses of pod formation, the lopac1280
and tpims combinatorial libraries were screened. no hits
were identified among the 1280 pharmacologically active
lopac1280 compounds, possibly providing evidence that the
Hcs assay is highly specific and does not suffer from promis-
cuous reactivity. screening of the tpims combinatorial librar-
ies, which consist of mixtures representing 5,287,896
compounds and several million peptides, revealed a bioactive
n-methyl triamine mixture. Further sar evaluations where
library deconvolution was performed by the positional scan-
ning method revealed a clear sar and thus validated the Hcs
assay for detection of pod-inducing compounds. Furthermore,
n-methyl triamines that were active in the pod assay did not
affect the distribution of control proteins, H2aX and chk1,
some types of dna damage have been shown to induce the
formation of pml-type bodies that are thought to be functionally
FIG. 4. pml oncogenic domain (pod)–inducing n-methyl triamines
do not induce dna damage. Hela cells were treated for 12 h with
interferon-γ (iFn-γ; 4 u/µl), dmso (0.1%), dimethylformamide
(dmF; 0.1%), cisplatin (25 µm), staurosporine (sts; 25 nm), etoposide
(25 µm), or 1422 compounds (n-methyl triamines; 10 µm). cells were
then immunostained for pods (mouse monoclonal antihuman pml,
alexa Fluor 488 chicken antimouse antibodies), phospho-H2aX (rabbit
polyclonal antihuman phospho-H2aX-ser139, alexa Fluor 568 goat
antirabbit antibodies), or phospho-chk1 (rabbit polyclonal antihuman
phospho-chk1-ser317, alexa Fluor 568 goat antirabbit antibodies).
pod-positive nuclei (%) were quantified as previously described.
p-H2aX and p-chk1 nuclear staining intensity was quantified using
cytoshop. mean and standard deviation are shown (n = 4 wells/condition,
>200 cells imaged and quantified per well).
HcS Assay for Pod Activators
Journal of Biomolecular Screening 16(2); 2011 www.slas.org 257
different from the tumor-suppressive and antiviral pods men-
tioned in the current study.25-29 thus, the Hcs counterscreen
assays described here eliminate dna-damaging compounds
from further consideration.
active compounds such as the n-methyl triamines could
potentially induce pods via several different mechanisms. First,
iFn-α, -β, and -γ induce pod formation by upregulating pml
and various pod-associated proteins.9 thus, compounds might
induce interferon production, a mechanism that can be readily
determined by measuring interferon elaboration into culture
supernatants (e.g., using immunoassays) or by testing activation
of interferon-inducible reporter genes (e.g., isge-luciferase).
second, arsenicals have been shown to activate pml by directly
conjugating cysteines within the zinc fingers of the pml rBcc
domain,30 and thus compounds that covalently modify these sites
define an additional mechanism for pod activation. third, con-
jugation of pml by the ubiquitin-like protein sumo is required
for pod formation.31 Hence, compounds that affect sumoylation
represent another potential mechanism, which might include, for
instance, inhibitors of the proteases responsible for de-sumoyla-
tion. Finally, pml binding proteins such as daxx are regulated
by phosphorylation.32 the protein kinase Zipk, for example, has
been shown to be required for interferon to induce pod forma-
tion.28 thus, compounds that influence the relevant kinases or
phosphatases represent another potential class of pod activators
that might be revealed by our Hcs assay.
Because of the role of pods in tumor suppression and host
defense against viruses, pod-inducing compounds may have
relevance to a wide variety of cancers and infectious diseases.
arsenicals are already being used to treat leukemias (such as
apl) and multiple myeloma.2,16 in addition, adenoviruses, her-
pes simplex virus–1, human cytomegalovirus, epstein-Barr
virus, papillomavirus, hepatitis d virus, human t-lymphotropic
virus–1, lymphocytic choriomeningitis, and rabies viruses dis-
rupt pods,9 suggesting that pod-inducing compounds may
also promote antiviral states that could be therapeutically use-
ful. the Hcs assay described here enables high-throughput
screening (Hts) for compounds with potential medicinal activ-
ity based on induction of pods, thus providing a route to
chemical modulators of pml that accommodates the diversity
of cellular mechanisms responsible for pod regulation in a
manner unachievable with standard biochemical Hts assays.
We thank tessa siegfried and melanie Hanaii for assistance
with manuscript preparation, as well as drs. paul diaz and
satoshi ogasawara for technical assistance. support by dod
grant W81XWH-08-0574 and niH grant ca-55164.
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address correspondence to:
Dr. John C. Reed
Sanford-Burnham Medical Research Institute
10901 North Torrey Pines Road, La Jolla, CA 92037