5-Ethynyl-2′-deoxyuridine as a molecular probe of cell proliferation for high-content siRNA screening assay by "click" chemistry

Article abstract of DOI:10.1007/s11426-011-4398-4

Labelling and identification of proliferating cells is important for the study of physiological or pathological processes in high-content screening (HCS) assays. Here we describe ethynyl deoxyuridine (EdU) as a biomarker for the assessment of cell proliferation and clearly demonstrate the feasibility of the EdU-labelling method for use in HCS assays. EdU detection is highly robust, reproducible, technically simple, and well suited for automated segmentation, which provides an excellent alternative for setting up multiplexed HCS assays of siRNA, miRNA and small-molecule libraries.

Full text of DOI:10.1007/s11426-011-4398-4

SCIENCE CHINA
Chemistry
November 2011 Vol.54 No.11: 1702–1710
doi: 10.1007/s11426-011-4398-4
• ARTICLES •
· SPECIAL TOPIC · In Honor of the 80th Birthday of Professor Ronald Breslow
5-Ethynyl-2′-deoxyuridine as a molecular probe of cell
proliferation for high-content siRNA screening assay
by “click” chemistry‡
CHEN MiaoJuan1†, QU DeZhong1,3†, CHI WeiLin1, WANG Wei1,3, REN XiaoShuai1,4,
CONG ShuJie1,3, LIANG PeiZhou1,4, FENG ShiPeng5 & ZHANG BiLiang1,2*
1Lab of RNA Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. Guangzhou 510530, China
2State Key Laboratory of Respiratory Diseases; Guangzhou Institute of Respiratory Diseases, Guangzhou 510182, China
3Graduate University of Chinese Academy of Sciences, Beijing 100049, China
4University of Science and Technology of China, School of Life Sciences, Hefei 230026, China
5Guangzhou RiboBio Co., Ltd., Guangzhou Science Park, Guangzhou 510663, China
Received July 31, 2011; accepted August 16, 2011; published online October 11, 2011
Labelling and identification of proliferating cells is important for the study of physiological or pathological processes in
high-content screening (HCS) assays. Here we describe ethynyl deoxyuridine (EdU) as a biomarker for the assessment of cell
proliferation and clearly demonstrate the feasibility of the EdU-labelling method for use in HCS assays. EdU detection is
highly robust, reproducible, technically simple, and well suited for automated segmentation, which provides an excellent al-
ternative for setting up multiplexed HCS assays of siRNA, miRNA and small-molecule libraries.
molecular probe, siRNA, miRNA, “click” chemistry
alkynyl group of EdU can react with fluorescent azide in a
1 Introduction
Cu(I)-catalysed [32] cycloaddition, known as a “click”
reaction, enabling detection of EdU incorporation into
cells by fluorescence microscopy or fluorescence-activated
cell sorting. Few groups have reported the use of EdU in
HCS. In this study, we investigated the application of EdU
in multiplexed HCS assays.
High-content screening (HCS) is defined as multiplexed
functional screening based on imaging multiple targets in
the physiologic context of intact cells by extraction of
multicolour fluorescence information [1], which has
played a key role in cell proliferation and functional stud-
ies. Labelling, detection or quantification of either prolif-
erating cells or cells in the S-phase of cell cycle progres-
sion is important in order to study physiological or patho-
logical processes involved in cell proliferation using HCS.
Recently, 5-ethynyl-2′-deoxyuridine (EdU), a new, non-
radioactive thymidine analogue, was developed by replac-
ing the methyl group at the 5-position of thymidine with
an alkynyl group and was shown to readily incorporate
into DNA during cell proliferation [2, 3]. The terminal
2 Materials and method
2.1 Chemicals
EdU was synthesized from 5-iodo-deoxycytidine and trime-
thyl-silylacetylene by the Sonogashira coupling reaction
(Figure 1(b)), as previously described [4]. The identity and
purity of EdU was confirmed by TLC, NMR, and mass spec-
trometric analysis. EdU, dissolved in DMSO at 100 mM, was
stored frozen. The fluorescent azide derivatives Cy3-azide
*Corresponding author (email: zhang_biliang@gibh.org)
†Equal contribution to this work.
‡Dedicated to Prof. Ronald Breslow on the occasion of his 80th birthday
© Science China Press and Springer-Verlag Berlin Heidelberg 2011
chem.scichina.com www.springerlink.com

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2.4 EdU/BrdU labelling of cultured cells
Before fixation of siRNA-transfected cells, either EdU or
BrdU was added to the culture media at a concentration of
100 μM for 2 h. After labelling, cells were fixed by incuba-
tion with 4% paraformaldehyde in PBS for 30 min, fol-
lowed by one wash with PBS.
2.5 EdU/BrdU staining
After paraformaldehyde fixation, EdU-labelled cells were
rinsed once with PBS and then stained by incubating for
30 min with 100 mM Tris (from 2 M stock, pH 8.5), 0.5
mM CuSO4, 10 μM fluorescent azide (from 10 mM stock
in DMSO), and 50 mM ascorbic acid (added last to the
mix from 0.5 M stock in water). The staining mix was
freshly prepared each time and was used to stain cells im-
mediately after the addition of ascorbate. After staining,
the cells in the 96-well microplate were washed several
times with PBS with 0.5% Triton X-100. EdU-stained
cells were counterstained with Hoechst or immunostained
using standard protocols. To detect cells with BrdU in-
corporation, cells were treated with 2 mol/L HCl for 30
mins at 37 °C, followed by washing with 0.1 mol/L sodi-
um borate. After washing with PBS, the standard immu-
nolabelling protocols were performed, as described in the
following section.
2.6 Immunofluorescence staining
EdU-stained cells were rinsed twice with PBS and blocked
in 10% normal goat serum. Cells were incubated with BrdU
or p65 antibody (Santa Cruz Biotechnology) in blocking
buffer for 1 h at room temperature or overnight at 4 °C. Af-
ter incubation with Alexa555-goat anti-mouse IgG antibody
or Alexa488-goat anti-rabbit IgG antibody (Invitrogen),
they were counterstained with Hoechst (Sigma). Cells were
then imaged and analyzed using a BD PathwayTM 855 sys-
tem equipped with a 20 × objective.
Figure 1 Structures of Cy3-azide, Cy5-azide and synthesis of 5-ethynyl-
2′-deoxycytidine (EdU). (a) The structures of Cy3/Cy5-azide and EdU. (b)
Synthesis of EdU, a thymidine analogue that carries a terminal alkyne
group instead of a methyl in the 5-position of the pyrimidine ring.
and Cy5-azide were synthesized as previously described [5].
2.2 Cell culture and siRNA transfection
Hela cells (ATCC, CCL-2) were grown in Dulbecco’s mod-
ified Eagle’s medium supplemented with 10% FBS in a
humidified incubator at 37 °C with 5% CO2. The growing
cells were harvested when the monolayer density reached
50%60%. The cells were passaged using trypsin and
seeded in growth medium onto 96-well microplates (BD
FalconTM, cat. no. 353219) at a concentration of 50008000
cells/well. After 24 h, cells were transfected with 50 nM
siRNA targeting the kinase genes or NT siRNA and
Lipofectamine 2000 (Invitrogen) for up to 48 h.
2.7 Automated image capture and analysis
A BD Pathway 855 system was used to screen for prolifera-
tion data. This system is based on an inverted epifluores-
cence microscope equipped with two mercury short arc
lamps. The instrument automatically focuses on samples
and scans fields by means of a motorised stage. The images
were acquired with an internal high-resolution Hamamatsu
ORCA ER CCD camera. Sixteen excitation and eight emis-
sion filters with independent dichroic mirrors were used to
acquire fluorescence in the Hoechst (380 ± 10 nm), green
(488 ± 10 nm), and red (548 ± 20 nm) channels. At least
2000 cells were analyzed in each well with a 20 × objective.
The images were automatically stored and analysed by At-
tovision software.
2.3 TNFα and MG132 treatment
NF-κB translocation was induced with 10 ng/mL TNFα
(R&D) for 30 min. To analyze inhibition of cell proliferation
and NF-κB translocation, cells were preincubated with 25
nM MG132 (Sigma) for 3.5 h prior to the addition of TNFα.

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Cells labelled in culture overnight with EdU showed very
3 Discussion and conclusions
intense nuclear staining after reaction with Cy3-azide or
Cy5-azide under Cu(I)-catalysed click reaction conditions
(Figure 1(c) and Figure 2). In contrast, cells not labelled
EdU and fluorescent azide derivatives were synthesized, as
described in the methods reported for this study (Figure 1).
Figure 2 Use of 5-ethynyl-2′-deoxycytidine (EdU) to label DNA in cells. (a) Schematic diagram of the click chemistry reaction for detecting EdU incor-
porated into cellular DNA. The terminal alkyne group reacts with a fluorescence-azide in the presence of catalytic amounts of Cu(I). (bd) Detection of EdU
incorporated into DNA of cultured HeLa cells by fluorescence microscopy. HeLa cells were incubated in media without EdU (b) or media supplemented
with 100 μM EdU (c, d). The cells were fixed and reacted with 10 μM Cy3-azide (b, c) or 10 μM Cy5-azide (d) for 30 min. The cells were then counter-
stained with Hoechst to reveal cellular DNA, washed, and imaged by fluorescence microscopy. Scale bar, 100 μm. (e) Quantification of the proliferation
index of HeLa cells by detection of EdU incorporation. The histogram represents the distribution of EdU-related fluorescence intensity. The total area is
integrated, and the area fractions on the left or right of the dotted line are reported as a percentage in the graph. Dot plots: the nuclear area of each single cell
is plotted versus the nuclear intensity. (f) Cell population analysis of EdU intensity versus DNA content of HeLa cells. Three different cell cycle phases,
G0/G1, S and G2/M, are separated by cell population analysis with EdU incorporation and DNA content.

Chen MJ, et al. Sci China Chem November (2011) Vol.54 No.11
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with EdU displayed no detectable staining if subjected to
the same fixation and staining conditions as EdU-labelled
cells (Figure 2(b)(d)). By detection of EdU incorporation,
we can analyze and quantify the proliferation index of cells.
Performing a segregated analysis, we obtained the fluores-
cence distribution histogram area responding to EdU incor-
poration and then applied a threshold to the fluorescence,
quantifying the area corresponding to EdU- positive cells
(the proliferation index) (Figure 2(e)). Combining this with
other independent cell cycle markers, we were also able to
Figure 3 EdU detection is well suited for setting up multiplexed high-content screening assays. (a) EdU labelling is compatible with the DNA dye
Hoechst. EdU was nuclear-specific, showing clear co-localisation with Hoechst, while BrdU reduced Hoechst binding. The BrdU micrograph was taken
using a 10-fold longer exposure than that of the EdU micrograph under identical digital camera settings. Scale bar, 100 μm. (b) EdU staining protocol
versus BrdU staining protocol for HCS plate assay. The entire protocol of EdU staining can be completed in 2 h, while the entire protocol of BrdU staining
required over 5 h.

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perform cell cycle analysis and quantify cell cycle popula-
tion. Our analysis successfully detected the cell population
in the S-phase of the cell cycle and mitotically blocked
cells with double DNA contents; it also effectively differ-
entiated between the dormant and proliferative states of
cells, which greatly aided in discovery of stage-specific
cell cycle inhibitors. As the results showed, the cell cycle
phases G0/G1, S and G2/M were separated by cell popula-
tion analysis with EdU incorporation and DNA content
(Figure 2(f)).
To demonstrate whether the EdU-labelling method was
better for labelling, detection and quantification of prolifer-
ating cells in HCS assays, we chose to compare it to the
conventional BrdU-labelling method. BrdU-labelling has
been useful for assessing cell proliferation, but it requires
harsh treatment of DNA, using concentrated hydrochloric
acid or mixtures of methanol and acetic acid to allow the
anti-BrdU antibody access to the incorporated BrdU subu-
nits. This results in the loss of the helical DNA structure,
which is required for cell cycle dye-binding, and destroys
many cellular antibody epitopes [68]. Compared to the
BrdU-labelling method, the advantages of EdU-labelling are
evident. The click reaction between the ethynyl group on
DNA and fluorescent azide does not require denaturation of
the specimen, which allows good structural preservation;
thus, EdU detection is compatible with other fluorescent
probes and protein staining, unlike BrdU-labelling. Hoechst
is a type of bisbenzimidazole dye that binds to DNA, which
is commonly used in the laboratory to analyze and quantify
DNA in live or fixed cells. We observed that EdU was
compatible with the DNA intercalating dye Hoechst, while
the harsh staining conditions of BrdU staining reduced
Hoechst binding (Figure 3(a)). These results suggested that
the application of EdU enabled simultaneous quantitative
monitoring of different cellular targets, indicating that the
application of EdU allows for setting up multiplexed high-
content assays. Moreover, this characteristic of EdU con-
tributes to automatic segmentation. It is known that HCS
uses automated microscopy to collect images of cultured
cells. The images are subjected to segmentation algorithms
to identify cells or sub-cellular structures and quantitate
their morphology (such as fluorescence intensity or object
shape, size and texture) from hundreds to millions of indi-
vidual cells. The automated segmentation often begins by
extracting nuclei from the DNA channel and then uses the
results as the initialisation for the segmentation of cells
from other channels; therefore, nuclei must be accurately
segmented for further analysis [9, 10]. In BrdU stained
cells, Hoechst distribution is both cytoplasmic and nuclear,
so some parts of the cytoplasm were inaccurately labelled as
nuclei after the initial segmentation (Figure 4(a)). This dis-
torted downstream analyses of cellular features derived
Figure 4 EdU contribution to performance of automatic segmentation. (a) Segmentation of nuclei in EdU stained cells and BrdU stained cells. After the
initial segmentation, some parts of the cytoplasm were labelled as nuclei in BrdU stained cells, while this is absent in EdU stained cells. Arrows denote the
segmented cells. (b) The effect of nuclear segmentation on the analysis of nuclear size corresponding to the data in (a).

Chen MJ, et al. Sci China Chem November (2011) Vol.54 No.11
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from nuclei segmentation, for example, the average size
range of the nucleus. The average size of the nucleus was
larger in BrdU stained cells compared to that of vehi-
cle-treated cells (Figure 4(b)), but this did not occur in EdU
stained cells (Figure 4). This finding indicates that the use
of EdU improves HCS data quality by reducing segmenta-
tion errors.
In addition, we determined that EdU staining has a
signal-to-noise ratio (S/N) superior to that of BrdU staining.
The S/N was determined from assays performed in 10
96-well plates and calculated based on the equation: S/N=
(mean signal–mean background)/standard deviation of
background. Compared to the observed S/N of 3:1 for BrdU
staining, EdU staining exhibited the robust S/N of 9:1. We
also found that technical simplicity is improved by reducing
the number of steps and thereby decreasing technician time
in the EdU staining protocol (Figure 3(b)), which is im-
portant in HCS assays. When developing an assay to test the
effectiveness of various drugs, much work is done to quan-
tify how well the assay works. The Z′ factor is a dimen-
sionless, simple statistical characteristic for each HCS assay,
which provides a useful tool for comparison and evaluation
of the quality of assays, and can be utilized in assay optimi-
zation and validation. The Z′ factor indicates how well sep-
Figure 5 The application of the EdU-labelling method in high-content siRNA screening. (a) Whole plate image and intensity cluster of the red channel
(EdU). (b) Analysis of the cell proliferation index by detection of EdU incorporation. Error bars represent standard error of measurement. (c) List of enhanc-
er and inhibitor siRNAs. Enhancer siRNAs are targeting genes that block cell cycle progression. Inhibitor siRNAs are targeting genes required for cell cycle
progression. The effects of inhibitor siRNAs on cell cycle progression are also shown in the list.

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arated the positive and negative controls are. Z′ factor > 0 is
potentially screenable while 1 > Z′ factor ≧ 0.5 is consid-
ered an excellent assay. Therefore, to further determine the
suitability of the EdU-labelling method in HCS, we calcu-
lated the Z′ factor from both means of measurements and
standard deviation from measurements on vehicle- and
EdU-treated cells. The results showed that the Z′ factor
ranged from 0.6 to 0.8, suggesting that EdU detection offers
a robust method to assess cell proliferation using HCS.
Taken together, these observations indicate that EdU is an
excellent alternative for the identification of proliferating
cells in HCS assays.
To confirm the applicability of the EdU-labelling method
in HCS assays, we chose 30 chemically synthesized and
validated siRNAs targeting kinase genes known or predict-
ed to function in cell proliferation and then performed a
pilot high-content siRNA screening using EdU-labelling
(Figure 5(a)). We analyzed and quantified the proliferation-
index of HeLa cells by detection of EdU incorporation
(Figure 5(b)). As the results demonstrated, the proliferation
index obtained from cells transfected with ATM, MAP3K4,
BMPR2, MAP3K2 or non-targeting (NT) siRNAs was sig-
nificantly different, as confirmed by Student’s t-test, com-
pared to the control group (Figure 6(a, b) online). To ana-
lyze EdU incorporation and DNA content, we performed
cell population analysis, identified inhibitor and enhancer
siRNAs, and distinguished stage-specific cell cycle inhibi-
tors (Figure 5(c)). Enhancer siRNAs are targeting genes that
block cell cycle progression and result in enhancement of
cell proliferation, while inhibitor siRNAs are targeting
genes required for cell cycle progression and result in inhi-
bition of cell proliferation. For example, cells transfected
with MAP3K4, BMPR2, or MAP3K2 siRNA (Figure 6(c)).
BMPR2 siRNA induced S-phase arrest, and MAP3K4
Figure 6 Analysis of the proliferation index and cell cycle progression. (a) Images of Hela cells transfected with ATM, MAP3K4, BMPR2, MAP3K2 or
non-targeting (NT) siRNAs. Hela cells had incorporated EdU and were counterstained with Hoechst. (b) Analysis of the cell proliferation index correspond-
ing to the data in (a). * is presented as the mean ± S.D. with P < 0.05 as calculated by Student’s t-test. ** is presented as the mean ± S.D. with P < 0.001 as
calculated by student’s t-test. (c) Quantitation of cell cycle population in G0/G1, S, and G2/M phases. The cells were transfected with siRNA targeting
BMPR2, MAP3K4 or MAP3K2 and NT siRNAs, respectively.

Chen MJ, et al. Sci China Chem November (2011) Vol.54 No.11
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siRNA caused arrest in the G0/G1 phase, while MAP3K2
siRNA promoted cell proliferation, suggesting that BMPR2
and MAP3K4 are required for cell cycle progression, but
MAP3K2 blocks cell cycle progression. These results indi-
cate that EdU detection provides an easy-to-use method for
labelling, detection, and quantification of both proliferating
cells and cells in the S-phase of cell cycle progression in
siRNA-based HCS.
clear co-localisation with Hoechst. Under mild conditions,
EdU staining had no effect on p65 protein staining, identi-
fied by immunolabelling with anti-p65 antibodies (Figure
8). To determine the suitability of this assay as a screening
tool, the Z’ factor was calculated for two targets from the
means and standard deviations of measurements on vehi-
cle- and TNFα-treated cells. The results showed Z′ values
of 0.61 (EdU) and 0.65 (NF-κB p65), suggesting it is a ro-
bust assay for concurrent detection of EdU incorporation
and the distribution of NF-κB p65. MG132 is a potent,
cell-permeable, and selective proteasome inhibitor that
inhibits NF-κB activation by preventing IκB degradation
[13]. MG132 was used because, in addition to inhibiting
TNFα-stimulated NF-κB p65 nuclear accumulation, it sig-
nificantly suppressed the proliferation of cells (Figure
9(ac)). These results confirmed that EdU is well suited for
use in multiplexed HCS assay based on imaging of multi-
ple targets.
Here we describe the use of EdU as a biomarker for as-
sessment of cell proliferation and clearly demonstrate the
feasibility of the EdU-labelling method in HCS assays. EdU
detection is highly robust, reproducible, technically simple,
and suitable for HCS assays. In fact, in our laboratory, the
EdU-labelling method has replaced the BrdU-labelling
method in HCS assays and has been used to perform siRNA,
miRNA and small-molecule library screenings. EdU detec-
tion is shown to be an excellent method for the measure-
ment of cell proliferation in HCS assays and could be read-
ily extended to many other applications.
To further investigate the use of EdU in HCS assays, we
set up a cell-based multiplex fluorescence imaging assay
permitting concurrent detection and quantification of DNA
content, EdU incorporation and the distribution of nuclear
factor-κB (NF-κB) p65/RelA between the cytoplasm and
the nucleus. This is a useful tool to confirm genes required
for the NF-κB pathway and have an effect on cell prolifera-
tion. NF-κB transcription factors play a key role in many
physiological processes, such as the innate and adaptive
immune responses, cell proliferation, cell death, and in-
flammation [11, 12]. Signal transduction by NF-κB p65 is
dependent on its translocation from the cytosol to the nu-
cleus, and fluorescence immunolabelling was used to mon-
itor changes in its cellular distribution. A typical NF-κB p65
distribution pattern was observed, with the vast majority of
the transcription factor present throughout the cytosolic
compartment before stimulation, followed by considerable
NF-κB p65 nuclear translocation upon TNFα treatment
(Figure 7(a, b)). Then we simultaneously detected Hoechst,
EdU and NF-κB p65. The results showed that the fluores-
cent azide probe for EdU was nuclear-specific, displaying
Figure 7 Time course of the translocation of NF-κB p65 to the nucleus of Hela cells after TNFα stimulation. (a) Hela cells were induced with 10 ng/mL
TNFα for the times indicated, and p65 was visualised by indirect immunofluorescence using BD pathway 855. (b) Quantitation of cells showing p65 trans-
location to the nucleus at the indicated time after TNFα stimulation.
Figure 8 Concurrent detection of EdU and the distribution of NF-κB p65. After 2-h exposure to EdU, Hela cells had incorporated EdU (red) and were
counterstained with Hoechst (blue) and anti-p65 (green).

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Figure 9 MG132 blocks NF-κB p65 nuclear accumulation and cell proliferation. (a) After 1.5-h exposure to EdU, Hela cells were incubated with vehicle
or 10 ng/mL TNFα for 30 min. Cells were pretreated with MG132 for 3.5 h before TNFα treatment. MG132 was kept in the medium before fixation. Fol-
lowing fixation, permeabilisation and immunolabelling, cells were imaged using BD pathway 855. (b) Cells in (a) were categorised as nuclear (p65 predom-
inantly in the nucleus) or cytoplasmic (p65 predominantly in the cytoplasm) based on the p65 immunostaining pattern. (c) Cell proliferation was quantified
by detection of EdU incorporation corresponding to the data in (a). ** is presented as the mean ± S.D. with P < 0.001, as calculated by student’s t-test. ## is
presented as the mean ± S.D. with P > 0.05, as calculated by Student’s t-test.
This research was supported by the National Natural Science Foundation
of China (30870535 and 90913017) (B. Zhang) and Introduced Innovative
R&D Team Program of Guangdong Province (Gene Silencing Technology
and Therapeutics).
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