NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells

Abstract

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol, however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma, BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human cells.

Full text

NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to
the Mitotic Spindle in Human Cells
David Siegel*, Jadwiga K. Kepa, David Ross
Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado,
United States of America
Abstract
NAD(P)H:quinone oxidoreductase 1 (NQO1) is an FAD containing quinone reductase that catalyzes the 2-electron reduction
of a broad range of quinones. The 2-electron reduction of quinones to hydroquinones by NQO1 is believed to be a
detoxification process since this reaction bypasses the formation of the highly reactive semiquinone. NQO1 is expressed at
high levels in normal epithelium, endothelium and adipocytes as well as in many human solid tumors. In addition to its
function as a quinone reductase NQO1 has been shown to reduce superoxide and regulate the 20 S proteasomal
degradation of proteins including p53. Biochemical studies have indicated that NQO1 is primarily located in the cytosol,
however, lower levels of NQO1 have also been found in the nucleus. In these studies we demonstrate using
immunocytochemistry and confocal imaging that NQO1 was found associated with mitotic spindles in cells undergoing
division. The association of NQO1 with the mitotic spindles was observed in many different human cell lines including
nontransformed cells (astrocytes, HUVEC) immortalized cell lines (HBMEC, 16HBE) and cancer (pancreatic adenocarcinoma,
BXPC3). Confocal analysis of double-labeling experiments demonstrated co-localization of NQO1with alpha-tubulin in
mitotic spindles. In studies with BxPc-3 human pancreatic cancer cells the association of NQO1 with mitotic spindles
appeared to be unchanged in the presence of NQO1 inhibitors ES936 or dicoumarol suggesting that NQO1 can associate
with the mitotic spindle and still retain catalytic activity. Analysis of archival human squamous lung carcinoma tissue
immunostained for NQO1 demonstrated positive staining for NQO1 in the spindles of mitotic cells. The purpose of this
study is to demonstrate for the first time the association of the quinone reductase NQO1 with the mitotic spindle in human
cells.
Citation: Siegel D, Kepa JK, Ross D (2012) NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Localizes to the Mitotic Spindle in Human Cells. PLoS ONE 7(9): e44861.
doi:10.1371/journal.pone.0044861
Editor: Stefan Strack, University of Iowa, United States of America
Received April 12, 2012; Accepted August 8, 2012; Published September 11, 2012
Copyright: ß2012 Siegel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grant R01ES108943. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: DS, DR and the University of Colorado are the licensor for the anti-NQO1 monoclonal antibody (clone A180). This does not alter the
authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: david.siegel@ucdenver.edu
Introduction
NAD(P)H:quinone oxidoreductase 1 (NQO1, DT-diaphorase,
EC 1.6.99.2) is a homodimeric flavoprotein that utilizes either
NADH or NADPH and catalyzes the 2-electron reduction of a
broad range of substrates most notably quinones [1]. The two-
electron reduction of quinones to hydroquinones by NQO1 is
believed to be primarily a detoxification reaction since it bypasses
the formation of the highly reactive semiquinone [1]. In many
cases, however, the reduction of quinones by NQO1 results in the
formation cytotoxic hydroquinones and the bioactivation of
quinone prodrugs by NQO1 has been utilized as a strategy to
target NQO1-rich cancer cells [2].
In normal tissues, NQO1 is expressed at relatively high levels in
epithelial tissues, vascular endothelium and adipocytes while in
cancer, NQO1 is expressed at high levels in many solid tumors
including lung (NSCLC), breast and pancreatic [3,4]. In humans,
the NQO1*2 polymorphism plays a major role in governing basal
protein levels of NQO1 [5]. The NQO1*2 polymorphism results
in a proline to serine amino acid substitution at position 187 in
NQO1 and this mutant protein undergoes rapid polyubiquitina-
tion by the E3 ubiquitin ligase STUB1/CHIP with subsequent
proteasomal degradation [6,7]. Individuals homozygous for the
NQO1*2 polymorphism are NQO1 null, while intermediate levels
of NQO1 protein are found in individuals with the heterozygous
genotype [5]. NQO1 is under transcriptional regulation by the
Keap1/NRF2 pathway and upregulation of NQO1 mRNA or
protein has been used extensively as a biomarker for NRF2
activation [8,9]. Upregulation of NQO1 may protect the cell from
oxidative damage due to the ability of NQO1 to reduce
superoxide to hydrogen peroxide and generate antioxidant forms
of vitamin E and co-enzyme Q [10,11,12].
In addition to its role as an antioxidant enzyme, NQO1 has
been shown to protect a wide range of proteins including p53 from
ubiquitin-independent 20 S proteasomal degradation [13,14]. The
protection of target proteins by NQO1 from 20 S proteasomal
degradation is dependent upon the redox state of NQO1 since
treatment with the NQO1 inhibitor dicoumarol has been shown to
enhance the 20 S proteasomal degradation of several target
proteins [13,14].
NQO1 is predominately located in the cytoplasm but low levels
of NQO1 have been found in the nucleus under normal conditions
[15]. Under conditions of stress NQO1 has been shown to migrate
to the nucleus where it is hypothesized that NQO1 may protect
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p53 against 20 S proteasomal degradation [16]. In experiments
designed to monitor the subcellular distribution of NQO1 in
human cells by confocal microscopy we discovered that NQO1
could also be found in association with the mitotic spindle.
Results
Immunofluorescent staining of the human pancreatic adeno-
carcinoma cell line BxPc-3 for NQO1 revealed that NQO1 is
located primarily in the cytosol of these cells (Fig. 1). However, in
BxPc-3 cells undergoing mitosis intense immunostaining for
NQO1 was observed on the mitotic spindles (Fig. 1). Our source
of anti-NQO1 monoclonal antibody for these studies was
conditioned tissue culture supernatant from mouse hybridoma
clone A180. In control studies, we utilized unconditioned media
(RPMI1640 containing 10% fetal bovine serum) in place of
hybridoma clone A180 supernatant and in these studies no
immunofluorescent staining of NQO1in BxPc3 cells or spindles
could be observed (Fig. S1). In addition, no observable difference
in immunostaining of NQO1 including spindles could be detected
in BxPc-3 cells between tissue culture supernatant from hybridoma
clone A180 or commercially prepared affinity purified mouse IgG
from hybridoma clone A180 (Fig S2).
To confirm that NQO1 was the target of monoclonal antibody
A180 we generated a BxPc-3 cell line stably expressing a
doxycycline-inducible anti-NQO1shRNA expression vector. The
growth of this genetically modified BxPc3 cell line in doxycyline
for 7 days resulted in near complete knockdown of NQO1 protein
expression as confirmed by immunoblotting for NQO1 (Fig 2). As
expected, doxycycline treated cells showed no detectable immu-
nostaining for NQO1 either in the cytosol or on mitotic spindles
(Fig 2). These data confirm that NQO1 is the target of monoclonal
antibody clone A180 and that the immunostaining of mitotic
spindles by anti-NQO1 antibodies from clone A180 is not
observed when NQO1 protein levels are knockdown.
To confirm co-localization of NQO1 with the microtubule
containing mitotic spindles, doubling-labeling experiments were
performed using antibodies against NQO1 and alpha-tubulin and
results from these studies confirmed the co-localization of NQO1
with alpha-tubulin in the mitoic spindles (Fig 3). The association of
NQO1 with the mitotic spindles could be observed throughout
mitosis from metaphase to cytokinesis (Fig. 3). In cells not
undergoing division no obvious co-localization of NQO1 with
alpha-tubulin was observed, (see Fig. 3 D–F) suggesting that the
association between NQO1 and microtubules is specific for mitotic
spindles. Treatment of BxPc-3 cells with the competitive inhibitor
dicoumarol or the mechanism-based inhibitor ES936 did not
prevent the association of NQO1 with mitotic spindles suggesting
that the association of NQO1 with the mitotic spindle is not
dependent upon the redox state of NQO1 (Fig. 4).
The immunofluorescent staining of NQO1 was performed in
many different human cell lines (Fig. 5) including cancer cell lines
(pancreatic), immortalized cell lines (HBMEC, 16HBE) and
primary cell lines (astrocytes, HUVEC). The human pancreatic
cancer cell line Panc-1, which has previously been genotyped as
homozygous for the NQO1*2 polymorphism [17] and character-
ized as NQO1 null, did not demonstrate detectable immunoflu-
orescent staining for NQO1 either in the cytosol or mitotic
spindles. Stable expression of NQO1 in the Panc-1 cell line (clone
panc-1/C5) resulted in significant staining for NQO1 in the
cytosol and mitotic spindles (Fig 6).
To determine if NQO1 could be found associated with mitotic
spindles in human tissues, we examined human NSCLC tissue
samples that had been immunostained for NQO1 in a previous
study [18]. Upon examination of squamous cell carcinomas
samples mitotic figures were located and immunostaining for
NQO1 was clearly visible on the mitotic spindles of these cells
(Fig 7). These data support our studies in cell culture that
demonstrate the association of NQO1 with mitotic spindles.
Discussion
These data demonstrate that the quinone oxidoreductase
NQO1 associates with the mitotic spindles and suggests that this
association with the mitotic spindle is independent of the redox
state of NQO1 since immunostaining for NQO1 on spindles was
not diminished in the presence of NQO1 inhibitors. While this is
the first report of localization of NQO1 with mitotic spindles,
previous studies using Xenopus egg extracts indentified NQO1 as
a target in a chemical screen of purine-based mitotic disrupters
[19]. In this study, diminutol (2-(1R-isopropyl-2-hydroxyethyla-
mino)-6-(3-aminophenylthio)-9-isopropylpurine), which prevents
microtubule polymerization, was also shown to inhibit NQO1
catalytic activity via competitive binding with NAD(P)H. These
same studies demonstrated immuno-depletion of NQO1 from
Xenopus egg extracts resulted in defects in spindle assembly [19].
Despite these findings that suggested NQO1 catalytic activity may
be linked to spindle assembly in Xenopus eggs, the authors did not
demonstrate localization of NQO1 with spindles [19]. Unlike
studies in Xenopus eggs, our studies in human cells have shown
Figure 1. Immunofluorescent staining of NQO1 in BxPc-3 human pancreatic cancer cells. Immunostaining for NQO1 reveals cytosolic
localization and intense staining of mitotic spindles in cells undergoing division (*).
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that NQO1 can be found associated with the mitotic spindles;
however, treatment of human cells with the NQO1 mechanism-
based inhibitor ES936, which resulted in .95% inhibition of
NQO1 catalytic activity, did not alter the growth rate of these cells
[20]. These data suggest that in human cells despite the binding of
NQO1 to the mitotic spindles NQO1 most likely does not play a
direct role in regulating mitosis. These observations are reinforced
by studies in human cell lines that lack NQO1 activity and protein
due to homozygous expression of the NQO1*2 polymorphism.
Many commonly used human cells lines (BE, MDA231, MDA468,
Panc-1, Caco-2) lack NQO1 activity and protein due to
homozygous expression of the NQO1*2 polymorphism and for
many of these cell lines isogenic pairs have been created following
the stable transfection of wild type NQO1. The reintroduction of
wild type NQO1 back into these cell lines did not significantly alter
the growth rate when compared to their NQO1 null isogenic
parental cells [21]. Taken together, these data imply that in
human cells NQO1 does not play a major role in regulating
mitosis but instead NQO1 may act in a protective role.
Figure 2. Knockdown of NQO1 eliminates immunostaining of NQO1 in BxPc-3 cells. Knockdown of NQO1 in BxPc-3 cells was performed
using a doxycyline- inducible anti-NQO1 shRNA methodologies. BxPc-3 cells were immunostained for NQO1 (green) in the absence (panel A) and
presence (panel B) of doxycyline pretreatment. DAPI staining was included in panel B to aid in the location of cells. Right panel: immunoblot
demonstrating near complete knockdown of NQO1 protein in the presence of doxycycline. Arrows indicate mitotic cells.
doi:10.1371/journal.pone.0044861.g002
Figure 3. Co-localization of NQO1 and alpha-tubulin in mitotic spindles and midbody region in BxPc-3 human pancreatic cancer
cells. Triple immunofluorescent staining for NQO1 (red), alpha-tubulin (green) and nuclei (blue). Association of NQO1 with spindles can be seen in
different stages of mitosis including metaphase (*), telophase (**) and the midbody region (arrow) during cytokinesis.
doi:10.1371/journal.pone.0044861.g003
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The ability of NQO1 to associate with the mitotic spindles in
human cells while still maintaining catalytic activity has implica-
tions for both quinone detoxification and activation reactions in
close proximity to critical mitotic apparatus. The reduction of
quinones by NQO1 could result in protection of vulnerable
microtubules from attack by electrophilic quinones. The benzene
metabolite p-benzoquinone has been shown in cell free systems to
react readily with sulfhydryl groups on microtubule proteins and
inhibit assembly into microtubules [22]. Alternatively, NQO1
could be the source of high levels of reactive oxygen species due to
redox cycling of unstable hydroquinones formed in close proximity
to the mitotic apparatus.
An alternative catalytic function of NQO1 may be to aid in
DNA repair during mitosis through the generation of NAD
+
an
essential cofactor for proteins including the family of poly (ADP
ribose) polymerase (PARP) enzymes and the sirtuins. PARP
enzymes have been shown to play an important role in the repair
of single strand DNA breaks [23] while SIRT6 has been proposed
to aid in DNA repair under conditions of oxidative stress by
binding directly to PARP1 and stimulating PARP1 poly-ADP-
ribosylase activity [24].
From our studies it is unclear whether NQO1 binds directly to
microtubules in mitotic spindles or binds to a protein complex that
associates with the mitotic spindles. NQO1 has many character-
istics of a microtubule binding protein including a net positive
charge and multiple domains containing clusters of positively
charged amino acids [25,26,27]. Alternatively, NQO1 could be
binding to another protein or protein complex that is tethered to
the mitotic spindle. In addition to its major role in quinone
metabolism NQO1 has emerged as a regulator of 20 S
proteasomal degradation [28]. A growing number of proteins
have been shown to be protected from ubiquitin-independent 20 S
proteasome degradation by NQO1 including p53, p63, p73 and
ornithine decarboxylase [14,29,30,31]. Interestingly, immunoflu-
orescence studies have previously demonstrated co-localization of
p53 with centrosomes and mitotic spindles in human cell lines [32]
and co-localization of the 20 S proteasome to centrosomes and
mitotic spindles in rat Schwann cells [33]. Whether NQO1
interacts with these proteins on the spindle to facilitate in the
stabilization and redistribution of selected proteins such as p53
during mitosis is currently under investigation. Recently, it has
been shown that persistent expression of NQO1 via p62-mediated
Nrf2 activation facilitated p53-dependent mitotic catastrophe
supporting a role for NQO1 in the stabilization of p53 during
mitosis [34].
In summary, these data report the association of NQO1 with
mitotic spindles in human cells using immunofluorescent staining
and suggest that the association of NQO1 with spindles may
involve protection against electrophic quinones, aid in the
generation of oxidized pyridine nucleotides or shield selected
proteins from 20 S degradation.
Materials and Methods
Reagents
Anti-NQO1 mouse monoclonal antibody (IgG
1
, clone A180)
was developed in our laboratory against full-length recombinant
human NQO1 protein and hybridoma supernatant (RPMI1640
containing 10% fetal bovine serum) from clone A180 was used
for these studies (dilution 1:5–1:20). Affinity purified anti-
NQO1 monoclonal antibody from clone A180 was obtained
commercially from Cell Signaling Technologies (Danvers, MA)
and used at a dilution of 1:200. Anti-alpha tubulin rabbit
polyclonal antibody was purchased from Abcam, (Cambridge
MA, Cat#15246, dilution 1:10,000). DyLight 488 and DyLight
544 conjugated goat anti-mouse IgG and DyLight 488
conjugated goat anti-rabbit IgG antibodies were purchased
from Jackson ImmunoResearch Laboratories (West Grove PA).
49,6-diamidino-2-phenylindole (DAPI,) dicoumarol and doxy-
cycline were obtained from Sigma (St. Louis MO). The NQO1
mechanism-based inhibitor ES936 (5-methoxy-1,2-dimethyl-3-
[(4-nitrophenoxy)methyl]indole-4,7-dione) was synthesized in
the laboratory of Dr. Christopher J. Moody, School of
Chemistry, University of Nottingham, U.K [35].
Cell Lines and Tissues
BXPC3 and Panc-1 human pancreatic cancer cell lines were
obtained from ATCC (Manassas, VA) and grown in RPMI 1640
containing 5% (v/v) fetal bovine serum and 1% (v/v) penicillin/
streptomycin. Panc-1 cells have previously been genotyped as
homozygous for the NQO1*2 polymorphism and are NQO1null
[17]. Panc-1/C5 cells stably express wildtype NQO1 and were
created from parental Panc-1 cells as described [17]. Primary
cultures of human umbilical cord vein endothelial cells (HUVEC)
Figure 4. NQO1 inhibitors do not prevent binding of NQO1 to mitotic spindles. Immunostaining for NQO1 (red) in BXPC3 cells pretreated
with DMSO (panel A), ES936 (500 nM, panel B) or dicoumarol (50mM, panel C). Cells were pretreated with inhibitors 2 hr before immunostaining.
Arrows indicate mitotic cells.
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were obtained from Cell Applications (San Diego, CA) and grown
in Endothelial Cell Growth Media (Cell Applications). Primary
human astrocytes were obtained from ScienCell Research Labs
(Carlsbad, CA) and grown in Astrocyte Medium (ScienCell
Research Labs). Immortalized human bone marrow endothelial
cells (HBMEC) were provided by Dr. B.B. Weksler (Cornell
University) and grown as previously described [36]. Immortalized
human bronchial epithelial cells [37] (16HBE) were provided by
Dr. Brian Day (National Jewish Health, Denver CO) and grown in
RPMI 1640 containing 5% fetal bovine serum and 1% penicillin/
Figure 5. Co-localization of NQO1 and alpha-tubulin with mitotic spindles in human cells. Triple immunofluorescent staining for NQO1
(red, panel A); alpha-tubulin (green, panel B); merged (with DAPI,panel C).
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streptomycin. All cells were maintained as monolayers at 37uCin
5% CO
2
.
For immunocytochemical studies all cells were plated onto glass
coverslips (18 mm60.15 mm circle, Fisher Scientific) in 100 mm
tissue cultures dishes 3 days prior to analysis. For studies using
NQO1 inhibitors ES936 and dicoumarol cells were incubated in
fresh media (2 ml) containing inhibitors (500 nM ES936 or 50 mM
dicoumarol) for 2 hr. The generation of a doxycycline-inducible
NQO1 knock-down cell line was performed using a TRIPZ
inducible shRNA plasmid for human NQO1 (Open Biosystems,
Lafayette, CO). To create a stable knockdown cell line, 2610
6
BxPc3 cells were electroporated with 20 ug of TRIPZ inducible
shRNA plasmid for human NQO1 and transfected cells were
selected in normal culture media containing 3 mg/ml puromycin.
The antibiotic resistant cells were induced with 300 ng/ml
doxycyclin for 48 h and then cells were sorted for turbo red
fluorescence (553excitation/574emission) using the Legacy MoFlo
sorter (University of Colorado Cancer Center). Sorted cells were
maintained in complete media containing 500 ng/ml doxycycline
and knockdown of NQO1 was confirmed by immunoblot analysis.
Coincubation studies with Panc-1 cells and Panc-1/C5 cells
were performed using the following method. Panc-1 cells (1610
6
)
Figure 6. Co-localization of NQO1 with mitotic spindles is not observed in cells homozygous for the NQO1*2 polymorphism. Panc-1
cells which are homozygous for the NQO1*2 polymorphism (NQO1 null), were prelabeled with cell tracker green then mixed with unlabled Panc-1/C5
cells which stably express wild type hNQO1. Following a 24hr coincubation period cells were fixed and immunostained for NQO1 followed by
confocal microscopy. Cells were searched until a mitotic Panc-1 cell (green) and a mitotic Panc-1/C5 cell (non-staining) were observed in the same
field. Panels A and C, data was collected using 488nm excitation wavelength for cell tracker green fluorescence (green Panc-1 cells). Panels B and D,
data was collected using 561nm excitation wavelength (red/NQO1). Co-localization of NQO1 to the mitotic spindles was not observed in NQO1-null
Panc-1 cells (green) while co-localization of NQO1 to the mitotic spindle was seen in NQO1 overexpressing Panc-1/C5 cells.
doi:10.1371/journal.pone.0044861.g006
Figure 7. NQO1 localizes to mitotic spindles in human lung cancer cells. Immunohistochemical analysis of NQO1 (DAB, brown) in archival
formalin-fixed human squamous lung carcinoma tissue samples. Mitotic figures are visible due to their intense chromatin staining (hemotoxylin).
doi:10.1371/journal.pone.0044861.g007
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were treated with 5 mM Cell Tracker Green (Invitrogen, Carlsbad,
CA) in serum-free media in suspension at 37uC. After 1 hr Panc-1
cells were collected by centrifugation, washed in PBS and then
mixed with unlabeled Panc-1/C5 cells (1610
6
) and plated onto
glass coverslips in complete media. After 24 hr the coverslips were
fixed for immunocytochemisty as described below.
Achieved formalin fixed human squamous lung cancer tissue
was obtained from the University of Colorado Cancer Center and
immunostained for NQO1 as previously described [18].
Immunocytochemistry
Immunocytochemistry was performed on cell lines grown on
glass coverslips using the following methods. All steps were
performed at room temperature. Cells were washed in phosphate
buffered saline (PBS) then fixed in 3.7% (v/v) formaldehyde in
PBS for 12 min, rinsed with PBS, then permeabilize with 0.15%
(v/v) Triton-X100 in PBS for 10 min. Cells were then blocked in
RPMI1640 containing 5% fetal bovine serum for 1 hr at room
temperature. Coverslips were then transferred to a 6-well plate
(single coverslip/well) and primary antibodies diluted in TBST
(10 mM Tris-HCl, pH 8 containing 150 mM NaCl and 0.4% (v/
v) Tween-20), were added (2 ml/well). After 90 min the primary
antibodies were removed, the cells were gently rinsed with TBST,
and secondary antibodies (diluted in TBST) were then added
(2 ml/well) for 30 min. DAPI (1 mg/ml) was included with the
secondary antibody. After 30 min coverslips were rinsed in TBST,
briefly immersed in deionized/distilled H
2
0, inverted, and
mounted on to glass microscope slides using SuperMount
(Biogenex, San Ramon, CA). Stained cells were examined using
a Nikon TE2000E2 inverted microscope equipped with a C1-Plus
confocal system (lasers 402 nm, 488 nm, 514 nm, Nikon Instru-
ments, Melville, NY).
Supporting Information
Figure S1 No fluorescent immunostaining of mitotic
spindles in the absence of anti-NQO1 primary antibody.
When unconditioned RPMI1640 containing 10% fetal bovine
serum was substituted for conditioned hybridoma clone A180
supernatant no immunostaining of cells or mitotic spindles could
be observed. Panel A, acquired with confocal settings used for
Figure 1; Panel B, acquired with confocal settings greatly
enhanced for detection of Dylight 549 nm (red). Arrows indicate
mitotic cells.
(TIF)
Figure S2 NQO1 immunostaining in BxPc-3 cells using
hybridoma supernatant or purified antibody. Identical
immunostaining of NQO1 in BxPc-3 cells was observed using
either (A) hybridoma tissue culture media from clone A180 diluted
1:10 or (B) affinity purified mouse IgG from clone A180 (Cell
Signaling Technologies) diluted 1:200.
(TIF)
Author Contributions
Conceived and designed the experiments: DS JK DR. Performed the
experiments: DS. Analyzed the data: DS JK DR. Contributed reagents/
materials/analysis tools: JK. Wrote the paper: DS JK DR.
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