Hypoxia Reduces Arylsulfatase B Activity and Silencing
Arylsulfatase B Replicates and Mediates the Effects of
Sumit Bhattacharyya1,2, Joanne K. Tobacman1,2*
1Department of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America, 2Department of Medicine, Jesse Brown VA Medical Center, Chicago,
Illinois, United States of America
This report presents evidence of 1) a role for arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) in mediating
intracellular oxygen signaling; 2) replication between the effects of ARSB silencing and hypoxia on sulfated
glycosaminoglycan content, cellular redox status, and expression of hypoxia-associated genes; and 3) a mechanism
whereby changes in chondroitin-4-sulfation that follow either hypoxia or ARSB silencing can induce transcriptional changes
through galectin-3. ARSB removes 4-sulfate groups from the non-reducing end of chondroitin-4-sulfate and dermatan
sulfate and is required for their degradation. For activity, ARSB requires modification of a critical cysteine residue by the
formylglycine generating enzyme and by molecular oxygen. When primary human bronchial and human colonic epithelial
cells were exposed to 10% O261 h, ARSB activity declined by ,41% and ,30% from baseline, as nuclear hypoxia inducible
factor (HIF)-1a increased by ,53% and ,37%. When ARSB was silenced, nuclear HIF-1a increased by ,81% and ,61% from
baseline, and mRNA expression increased to 3.73 (60.34) times baseline. Inversely, ARSB overexpression reduced nuclear
HIF-1a by ,37% and ,54% from baseline in the epithelial cells. Hypoxia, like ARSB silencing, significantly increased the total
cellular sulfated glycosaminoglycans and chondroitin-4-sulfate (C4S) content. Both hypoxia and ARSB silencing had similar
effects on the cellular redox status and on mRNA expression of hypoxia-associated genes. Transcriptional effects of both
ARSB silencing and hypoxia may be mediated by reduction in galectin-3 binding to more highly sulfated C4S, since the
galectin-3 that co-immunoprecipitated with C4S declined and the nuclear galectin-3 increased following ARSB knockdown
Citation: Bhattacharyya S, Tobacman JK (2012) Hypoxia Reduces Arylsulfatase B Activity and Silencing Arylsulfatase B Replicates and Mediates the Effects of
Hypoxia. PLoS ONE 7(3): e33250. doi:10.1371/journal.pone.0033250
Editor: Suzannah Rutherford, Fred Hutchinson Cancer Research Center, United States of America
Received October 3, 2011; Accepted February 13, 2012; Published March 13, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: The sources of funding were VA Merit Review and Clinical and Translational Science Award (CTSA) UL1 RR029879. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Deficiency of the enzyme arylsulfatase B (ARSB; N-acetylga-
lactosamine-4-sulfatase) leads to the lysosomal storage disease
mucopolysaccharidosis (MPS) VI (Maroteaux-Lamy-Syndrome),
which is associated with accumulation of the sulfated glycosoami-
noglycans chondroitin-4-sulfate (C4S) and dermatan sulfate (DS).
In addition to lysosomal localization, ARSB is also present in the
cell membrane of epithelial and endothelial cells [1–6]. The
sulfatase enzymes are a family of enzymes that each have highly
specified chemical function, and ARSB removes the 4-sulfate
group from N-acetylgalactosamine-4-sulfate at the non-reducing
end of C4S and DS, and thereby can regulate the degradation of
these sulfated glycosaminoglycans (GAGs) [7–9]. The catalytic
activity of ARSB requires post-transformational modification of a
critical cysteine residue at position 91 by formylglycine modifica-
tion, involving the formylglycine-generating enzyme (FGE) and
molecular oxygen [10–14]. Other work has characterized the FGE
as sulfatase modifying factor (SUMF)1 and addressed its migration
from the endoplasmic reticulum, its secretion, and return to the
endoplasmic reticulum [15–17]. Crystallization of ARSB demon-
strated that the sterically favorable conformation of ARSB involves
participation of a calcium ion to which the sulfate group from the
C4 position of the N-acetylgalactosamine residue is bound [18,19].
The critical cysteine residue of ARSB is restored following
conversion by the FGE and molecular oxygen to a formylglycine
(oxo-alanine) intermediate [10–14]. Specific correction of ARSB
deficiency in MPS VI has been achieved by direct intravenous
administration of recombinant human ARSB produced in CHO
cells in which the post-translational formylglycine modification
Our recent reports have indicated that ARSB activity is reduced
in malignant colonic and mammary epithelial cells [1,3,22,23] and
in uncorrected cystic fibrosis bronchial epithelial cells .
Decline in ARSB activity has lead to reduced IL-8 secretion and
increased IL-8 sequestration by bronchial epithelial cells  and
increased cell-based kininogen and reduced bradykinin secretion
by normal rat kidney epithelial cells . Work of other
investigators has shown the importance of chondroitin-4-sulfation
in relation to attachment of malarial parasites to placental and
vascular cells [25,26], and the impact of chondroitin-4-sulfation on
glial scar formation following spinal cord injury . These studies
demonstrate that ARSB, due to its essential role in the degradation
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of chondroitin-4-sulfate, has profound effects on a wide range of
pathophysiological processes in multiple cell types and models.
The studies in this report were undertaken to better understand
how ARSB activity and chondroitin-4-sulfation could influence
such diverse and significant biological processes. In this report, the
similar effects of hypoxia and of ARSB silencing in human
bronchial and colonic epithelial cells in culture are presented for
the first time. Since molecular oxygen is required for the post-
translational modification of ARSB to its active form, we
hypothesized that ARSB activity might be reduced in a hypoxic
environment and that intracellular oxygen signaling might be
mediated by ARSB. We considered if ARSB silencing could
replicate the effects of oxygen on HIF-1a. Determinations of
nuclear HIF-1a levels and mRNA expression were performed and
supported the emerging concept that ARSB activation might serve
as a cellular rheostat to regulate molecular oxygen signaling. To
obtain additional evidence about the similarity of the effects of
hypoxia and ARSB, the impact of hypoxia on the expression of 84
genes in a hypoxia signaling PCR array and on the levels of
sulfated glycosaminoglycan (GAG) and chondroitin-4-sulfate (C4S)
levels were determined. Recognizing that a mechanism by which
the impact of ARSB effects on N-acetylgalactosamine-4-sulfation
could be translated into transcriptional events was required, we
proceeded to investigate the potential role of galectin-3 as an
intermediary between changes in chondroitin sulfation and gene
expression, based on the reported decline in galectin binding to
more highly sulfated chondroitin sulfate . Study findings that
follow support galectin-mediation of signaling from environmental
oxygen, through ARSB activation and chondroitin-4-sulfation, to
nuclear AP-1-activation and enhanced HIF-1a transcription.
Also of interest was the consideration of how ARSB silencing
and hypoxia might affect the impact of sulfate groups on cellular
redox status, since sulfate assimilation is a well-recognized
metabolic process in protists, plants, and yeast [29–31]. The
sulfate assimilation pathway comprises a series of redox reactions
by which sulfates are progressively reduced to sulfhydryls,
including reduced glutathione and cysteine. In addition, sulfate
groups participate in the sulfotransferase reactions that involve the
39-phosphoadenosine 59-phosphosulfate (PAPS), and thereby
recycle into sulfated glycosaminoglycan synthetic processes and
into sulfated storage substrates [32–36]. Measurements of total
cellular sulfhydryl content and of the ratio of reduced glutathione
to gluthatione disulfide were undertaken to assess if ARSB
silencing and hypoxia both produced similar changes in the
overall cellular redox status, consistent with inhibition of sulfate
assimilation and reduced sulfhydryl production.
The experiments that follow in this report consider how ARSB
mediates oxygen signaling; how changes in chondroitin-4-sulfation
lead to transcriptional effects through changes in galectin-3 binding
and galectin-3 nuclear translocation; and how the redox effects of
oxygen and ARSB may result from effects on sulfate assimilation in
human epithelial cells. We anticipate that further consideration of
the role of sulfate in cellular metabolism will lead to improved
understandings of the mechanisms that determine cell fate.
Hypoxia reduces ARSB activity in human epithelial cells
Since molecular oxygen is required for activation of ARSB, the
effect of a reduced oxygen environment on ARSB activity was
measured. Normal human bronchial epithelial cells (BEC) and
human colonic epithelial cells (NCM460 cells) were exposed to a
reduced oxygen environment (10%) for 15 minutes to 24 hours,
and ARSB activity was determined using the exogenous substrate
4-methylumbilliferyl sulfate. PO2declined 40–45% in the hypoxic
NCM460 cells compared to the normoxic control cells at 1 h and
4 h. Maximum lowering of ARSB activity occurred by 1 h,
declining to 57% (from 72.762.9 to 41.561.6 nmol/mg protein/
h) and to 67% (from 130.067.8 to 87.863.7 nmol/mg protein/h)
of the baseline activity, in the BEC (Fig. 1A) and NCM460 cells
(Fig. 1B), respectively (p,0.001; 1-way ANOVA with Tukey-
Kramer post-test). In contrast, activity of galactose-6-sulfatase
(GALNS), steroid sulfatase (STS), or arylsulfatase A (ARSA) did
not decline in the BEC or NCM460 cells following exposure to the
reduced oxygen environment.
ARSB silencing by siRNA produced greater declines in ARSB
activity than the 10% oxygen environment. ARSB declined 81%
(from 70.766.7 to 13.561.0 nmol/mg protein/h) in the BEC and
85% (from 129.266.1 to 19.862.7 nmol/mg protein/h) in the
NCM460 cells. The combination of hypoxia and ARSB silencing
by siRNA did not produce any further decline in the ARSB
activity than ARSB silencing alone in the BEC (Fig. 1C) or the
NCM460 cells (Fig. 1D). Return to normoxia following exposure
to hypoxia restored ARSB activity to baseline in the BEC (Fig. 1E)
and NCM460 cells (Fig. 1F). Overexpression of ARSB increased
activity to 175.669.4 nmol/mg protein/h in the BEC and to
230.4610.7 nmol/mg protein/h in the BEC.
To rule out effects of hypoxia on the sulfatase modifying factor
(SUMF)-1 as the explanation for reduced ARSB activity, SUMF-1
was measured in the BEC and NCM460 cells (Fig. 2A). SUMF-1
acts as a formylglycine modifying enzyme (FGE), converting the
critical cysteine residue in ARSB to a formylglycine. Hypoxia
produced no decline in SUMF-1 content in the BEC or NCM460
cells. Silencing SUMF-1 by siRNA (Fig. 2B) significantly reduced
the ARSB activity in the NCM460 cells (Fig. 2C) (p,0.001).
Maximum reduction in ARSB activity was achieved by the
combination of SUMF-1 silencing and hypoxia (p,0.001)
(Fig. 2C), consistent with requirements for molecular oxygen
and formylglycine modification for ARSB activity.
Hypoxia reduces total sulfated glycosaminoglycans and
To determine if hypoxia affected cellular sulfated glycosamino-
glycans (GAG) or chondroitin-4-sulfate (C4S) content, total cell-
associated sulfated GAGs and C4S were measured following
exposure of the BEC and the NCM460 cells to 10% O2 for
24 hours. Sulfatases, galactosidases, and chondroitinases are in-
volved in sulfated GAG degradation, but no effect of hypoxia on
GAG abundance was anticipated. However, in the hypoxic
condition, total sulfated GAGs increased from 12.360.5 mg/mg
proteinto 16.961.0 mg/mg protein in the BEC (p=0.002, unpaired
t-test, two-tailed) and from 14.0 mg/mg protein to 16.860.9 mg/mg
protein in the NCM460 cells (p=0.013, unpaired t-test, two-tailed)
(Fig. 3A). These increases were mainly attributable to increase in
C4S levels, and consistent with decline in ARSB activity leading to
inhibition of removal of sulfate groups and inhibition of subsequent
degradation. In the BEC, C4S increased from 6.760.3 to
10.460.1 mg/mg protein (p,0.001, unpaired t-test, two-tailed)
and from 7.560.4 mg/mg protein to 9.860.3 mg/mg protein in
the NCM460 cells (p=0.002, unpaired t-test, two-tailed) (Fig. 3B).
The hypoxia-induced increases in total sulfated GAG and C4S
content correlate with previously reported changes that followed
ARSB silencing and overexpression in the BEC and NCM460
cells and are proportionate to the hypoxia-induced decline in
ARSB activity. The cellular sulfated GAG and C4S content are
inversely and linearly related to the ARSB activity, including at
baseline, and following hypoxia, silencing, and overexpression in
the BEC and the NCM460 cells with highly significant inverse
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correlation (r=20.99) (Fig. 3C, 3D). Similar correlations
(r=20.99) were observed for C4S in the BEC (Fig. 3E) and in
the NCM460 cells (Fig. 3F) in relation to the ARSB activity.
ARSB silencing increases and ARSB overexpression
reduces the activation and expression of HIF-1a
To assess if some cellular effects of oxygen might be mediated by
changes in ARSB activity, measurements of HIF-1a following
ARSB silencing and overexpression were performed. When ARSB
was silenced by siRNA in the BEC, nuclear HIF-1a increased to
,181% of baseline. Following ARSB overexpression, nuclear
HIF-1a declined to ,46% of baseline (p,0.001) (Fig. 4A).
Similarly, nuclear HIF-1a increased following ARSB silencing in
the NCM460 cells, and ARSB overexpression produced significant
decline in HIF-1a (p,0.001) (Fig. 4B). The increases in activated,
nuclear HIF-1a in the BEC and the NCM460 cells following
ARSB silencing were similar at 1 and 4 hours, but the increases
were less at 24 hours (Fig. 4C, 4D). The increases in activated
HIF-1a were greater following ARSB silencing than from
exposure to 10% O2for 1–24 hr.
The combination of ARSB silencing and hypoxia had no
greater impact on stimulating HIF-1a than ARSB silencing alone
in the BEC (Fig. 4E) or in the NCM460 cells (Fig. 4F). The
relationship between nuclear HIF-1a and ARSB activity,
including at baseline and following hypoxia, silencing, and
overexpression, reveals a direct, inverse correlation between
ARSB activity and nuclear HIF-1a in the BEC (r=20.96) and
in the NCM460 cells (r=20.98) (Fig. 4G, 4H).
mRNA expression of HIF-1a in the BEC was determined by
PCR and indicated an increase to 2.560.2 times the control
following exposure to hypoxia (10% O264 h) and to 3.760.3
times the control siRNA following ARSB silencing 624 h
(p,0.001) (Fig. 4I).
Both hypoxia and ARSB silencing decrease the reduced
glutathione/glutathione disulfide ratio and total cellular
The unexpected replication by ARSB of the effects of oxygen on
HIF-1a suggested that ARSB might act to mediate effects of
oxygen through a broad range of cellular effects. We proceeded to
Figure 1. ARSB activity reduced by hypoxia in BEC and NCM460 cells. A. ARSB activity in the bronchial epithelial cells (BEC) was measured
following exposure to 10% oxygen environment for 0.25, 0.5, 0.75, 1, 4, and 24 hours. ARSB declined significantly in activity by 0.25 hours (p,0.001),
and remained at this level for 24 hours, in contrast to control cells under normoxic conditions. B. Similarly, following exposure to 10% O2, ARSB
activity declined in the NCM460 cells and was significantly reduced at 0.25 h (p,0.05). ARSB declined further by 30 minutes (p,0.01), reaching
maximum reduction by 0.75 h (p,0.001), and remained at this level for 24 h, in contrast to control cells. C. The combination of hypoxia (10%
O264 h) and ARSB silencing in the BEC produced no further decline than that achieved by ARSB silencing alone. D. Similarly, in the NCM460 cells, the
combination of ARSB silencing and hypoxia (10% O264 h) did not lead to further decline in the ARSB activity than that produced by ARSB siRNA
alone. E. In the BEC, return to normoxia for 4 h after hypoxia (10% O264 h) restored the baseline ARSB activity. F. In the NCM460 cells, return to
normoxia for 4 h after 4 h of 10% O2restored the baseline ARSB activity. [ARSB=arylsulfatase B; BEC=bronchial epithelial cell; N.D.=no difference].
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assess the impact of hypoxia and ARSB silencing on the overall
cellular redox status in the epithelial cells by examining the ratio of
reduced glutathione to glutathione disulfide (GSH/GSSG) and the
cellular sulfhydryl content.
In the human bronchial and colonic epithelial cells, both
exposure to the reduced oxygen environment (10% O264 h) and
ARSB silencing produced declines in the GSH/GSSG ratio
(Fig. 5A, 5B). In the BEC, the GSSG content increased to
0.6060.04 nmol/mg protein from 0.2460.02 nmol/mg protein,
with an associated decline in GSH. Similarly, in the NCM460
cells, the GSSG content increased to 0.4660.02 nmol/mg protein
from 0.2160.02 nmol/mg protein, and the GSH content declined
following exposure to hypoxia.
When ARSB was silenced, greater increases in GSSG in the
BEC (to 0.7260.07 nmol/mg protein) and in the NCM460 cells
(to 0.5960.04 nmol/mg protein) occurred, with greater declines
in GSH content and in the GSH/GSSG ratio. These effects
demonstrate similarities between the impact of hypoxia and ARSB
silencing on the cellular redox state, as manifested by changes in
GSH and G-S-S-G.
To further assess how changes in ARSB activity and in
oxygenation affected overall cellular redox status, measurements
of the total cellular sulfhydryl content, composed of protein-
associated and inorganic thiols, were also performed. ARSB
knockdown in the BEC and NCM460 cells produced marked
declines in the total cell thiol content (p,0.001), attributable to a
decline in the protein-associated sulfhydryls, since the declines in
the measured inorganic sulfhydryl content were not significant
(Fig. 6A, 6B). These findings are consistent with the declines in
the GSH/GSSG ratio. Exposure of the cells to the hypoxic
environment produced a smaller reduction in the total thiol
content than ARSB silencing, but proportionate to the hypoxia-
induced decline in the ARSB activity.
Hypoxia and ARSB silencing have similar effects on
expression of genes in hypoxia signaling array
Due to the unexpected and consistent similarities between the
effects of hypoxia and ARSB knockdown, the impact of hypoxia
and ARSB silencing on gene expression in a hypoxia signaling
PCR array was addressed to assess the extent of congruity between
the effects of hypoxia and ARSB knockdown. PCR was performed
using a hypoxia-signaling array that assayed expression of 84 genes
associated with hypoxia and 5 housekeeping genes. Cycle
threshold values were averaged using determinations from three
replicate arrays and corrected for housekeeping genes. Compar-
ison of the average cycle threshold values between the ARSB
silenced and hypoxic results demonstrated that in all cases the
direction (increase or decrease) and extent of change in expression
were similar between the effects of hypoxia and ARSB silencing, as
determined by average cycle threshold values, following correction
by the housekeeping genes (r=0.99; see Table S1) (Fig. 7). Of
the genes evaluated, six had significant p-values (p#0.05) and fold-
changes $1.6 (Table 1) following both hypoxia and ARSB
silencing, when compared to control or scrambled control siRNA,
respectively. The affected genes included: interleukin-6 (IL-6);
pentraxin-related gene (PTX3); collagen, type 1 alpha 1
(COL1A1); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3
(PLOD3); plasminogen activator, urokinase (PLAU); and heme
oxygenase 1 (HMOX1). Several other genes, including HIF-1a,
also had increased expression, but fold-change was less than 1.6.
Galectin-3 binding to C4S declined following hypoxia
and ARSB silencing
Multiple changes in gene expression followed both hypoxia or
ARSB silencing, but a molecular mechanism to explain these
effects was undetermined. The only mechanistic action of ARSB is
to remove sulfate groups from N-acetylgalactosamine-4-sulfate
residues, so transcriptional effects of ARSB silencing must be
related to the inhibition of this function. Preferential binding of
three members (galectins-3, -7, and -9) of a beta-galactoside-
binding protein family to desulfated vs. sulfated GAG has been
reported . We hypothesized that galectin-3 binding to C4S
might be reduced when ARSB activity was less and chondroitin-4-
sulfation was increased. Measurements of galectin-3 co-immuno-
precipitated with C4S were performed by ELISA following ARSB
silencing and hypoxia (10% O264 h). Galectin-3 declined from
,11.3 ng/mg protein to 4.660.2 ng/mg protein (p,0.001)
following ARSB silencing for 24 h (Fig. 8A) and to 6.960.3 ng/
mg protein (p,0.001) following hypoxia (Fig. 8B), consistent with
reduced galectin-3 binding to the more highly sulfated C4S. The
Figure 2. SUMF1 not modified by hypoxia in BEC or NCM460
cells. A. Protein expression of the sulfatase modifying factor (SUMF)-1
was not reduced by hypoxia (10% O264 h) in the BEC or the NCM460
cells. B. Knockdown by siRNA for SUMF-1 effectively reduced the
protein expression measured by ELISA in the NCM460 cells. C. ARSB
activity was significantly reduced by siRNA for SUMF-1 (p,0.001), and
additional reduction occurred in association with hypoxia (10% O264 h)
(p,0.001) in the NCM460 cells, consistent with a requirement for both
SUMF-1 and molecular oxygen for maximum ARSB activity. [ARSB=ar-
ylsulfatase B; BEC=bronchial epithelial cell].
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nuclear galectin-3 increased following both ARSB silencing
(Fig. 8C) and hypoxia (Fig. 8D) in the BEC.
Proposed mechanism for transcriptional effect of oxygen
and ARSB on HIF-1a by galectin-3 and AP-1
Galectin-3 was considered to be involved in the transcriptional
events that followed hypoxia and ARSB silencing, since its binding
to C4S and nuclear localization were significantly modified. The
effects of galectin-3 have been associated with interactions with AP-
1 components c-Jun and c-Fos, and AP-1 binding sites are present
on the HIF-1a promoter, suggesting that ARSB silencing and
hypoxia might induce transcriptional events through galectin-3 and
AP-1. ELISA assays demonstrated significant increases in nuclear c-
Jun(Fig.9A)andc-Fos(Fig. 9B) (p,0.001), suggestinga molecular
mechanism involving AP-1 and galectin-3 by which hypoxia and
the associated decline in ARSB activity and increase in chondroitin-
4-sulfation might regulate transcriptional activation of HIF-1a.
The study findings indicate that ARSB silencing replicates the
effects of hypoxia and are consistent with the requirement for
molecular oxygen to activate ARSB. Furthermore, the results
suggest that ARSB may mediate intracellular oxygen signaling
through effects on chondroitin-4- sulfation. Under normoxic
conditions, ARSB silencing increased HIF-1a activation and
expression, and ARSB overexpression reduced HIF-1a in human
bronchial and colonic epithelial cells, demonstrating the ability of
ARSB to regulate HIF-1a activation and expression. In addition to
similar effects on HIF-1a activation and expression, hypoxia and
ARSB silencing have similar effects, including 1) increase in
cellular sulfated GAG and C4S content, 2) decline in GSH/GSSG
ratio and total sulfhydryl level, 3) increase or decrease in mRNA
expression of 84 hypoxia-associated genes in a PCR array, 4)
increase in galectin-3 nuclear localization, and 5) AP-1 activation.
These effects suggest a central role for ARSB in the regulation of
vital cellular processes.
In addition to the findings that indicate which ARSB silencing
can mediate and replicate the effects of hypoxia, the study results
provide evidence to explain how the transcriptional effects of
hypoxia and ARSB silencing might be mediated. Reduced binding
of galectin-3 to C4S following reduction of ARSB activity was
demonstrated by decline in the amount of galectin-3 that co-
immunoprecipitated with C4S following hypoxia or ARSB
Figure 3. Total sGAG and C4S increased by hypoxia in BEC and NCM460 cells. A. In the BEC and the NCM460 cells, the total sGAG increased
significantly following 10% O2624 h (p=0.002, p=0.013 respectively, unpaired t-test, two tailed), consistent with an effect on ARSB function. B. The
majority of the increase in sGAG level following 10% O2624 h was attributable to the increases in C4S in the BEC and the NCM460 cells (p,0.001,
p=0.002, respectively). C, D. Highly significant inverse correlations were present between the sGAG content and the ARSB activity, using
measurements of sGAG and ARSB at baseline, following ARSB knockdown by siRNA, following hypoxia (10% O2624 h), and following ARSB
overexpression in the BEC (r=20.96) and NCM460 cells (r=20.999). E, F. Similar highly significant inverse correlations were present between the
C4S content and the ARSB activity in the BEC (r=20.95) and NCM460 cells (r=20.99). [sGAG=sulfated glycosaminoglycan; BEC=bronchial epithelial
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Figure 4. Nuclear HIF-1a levels were increased by ARSB silencing and reduced by ARSB overexpression in BEC and NCM460 cells,
and HIF-1a mRNA expression was increased by ARSB silencing in BEC. A. Activation of HIF-1a was significantly increased by silencing ARSB
and significantly reduced by overexpression of ARSB in the BEC (p,0.001). B. In the NMC460 cells, ARSB silencing significantly increased the
activation of HIF-1a and overexpression significantly reduced HIF-1a activation in the NCM460 cells (p,0.001). C. In the BEC, the effects of hypoxia
(10% O2) on HIF-1a activation were significant at 1 h (p,0.01), 4 h (p,0.001), and 24 h (p,0.01), with the maximum increase at 4 h. D. As in the BEC,
in the NCM460 cells, the effects of hypoxia (10% O2) on HIF-1a activation were significant at 1 h (p,0.01), 4 h (p,0.001) and 24 h (p,0.05), with the
maximum increase at 4 h. E. In the BEC, the combined effects of ARSB silencing and hypoxia (10% O2) for 24 h had no greater effect on activation of
HIF-1a than ARSB silencing alone. F. Similarly, in the NCM460 cells, the combined effects of ARSB silencing and hypoxia (10% O2) for 24 h had no
greater effect on the activation of HIF-1a than ARSB silencing alone. G, H. The ARSB activity at baseline, following ARSB overexpression, following
ARSB silencing by siRNA, and following hypoxia (10% O2)624 h were inversely correlated with the nuclear HIF-1a levels in the BEC and the NCM460
cells (r=20.96; r=20.98, respectively). I. HIF-1a mRNA expression increased to 2.5 (60.23) times the baseline following 10% oxygen64 h and to 3.73
(60.34) times the baseline following ARSB silencing by siRNA624 h (p,0.001) in the BEC. [ARSB=arylsulfatase B; BEC=bronchial epithelial cell;
HIF=hypoxia inducible factor].
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knockdown. Decline in binding to more highly sulfated C4S was
associated with increased nuclear galectin-3. Increases in nuclear
AP-1 components c-Fos and c-Jun were also shown following
ARSB silencing and hypoxia. Galectin-3 has been reported to
induce transcriptional effects on MUC2 in association with AP-1
in human colon cancer cells , and we propose that the effects
of hypoxia and ARSB silencing on the activation of HIF-1a may
be achieved through the AP-1 binding sites present in the HIF-1a
promoter in the presence of the increased nuclear galectin-3 .
Additional effects of ARSB, as well as of oxygen, may be mediated
through expression of HIF-1a, which has a broad repertoire of
known effects, including impact on cell proliferation and epithelial-
mesenchymal transition [39,40]. Galectin-3, like p53 and c-Myc,
has a nuclear localization motif and interacts with importins a and
b for nuclear translocation and has a wide range of effects on vital
cellular processes [41,42]. The binding of other galectins to C4S
may also be modified by changes in chondroitin sulfation when
ARSB activity is reduced .
Figure 10 is a schematic representation of the signaling in
normoxic and hypoxic pathways, mediated through changes in
ARSB activity. Transcriptional events following hypoxia may
proceed from reduced ARSB activity, thereby modulating
chondroitin-4-sulfation, galectin-3 binding to chondroitin-4-sul-
fate, galectin-3 nuclear translocation, and AP-1 activation.
Variation in the chondroitin-4-sulfation interaction with galectin-
3 affects the availability of galectin-3 for nuclear translocation and
subsequent transcriptional events involving interaction of galectin-
3 with AP-1. The promoters for IL-6, HMOX1, PLAU, COL1A1,
genes significantly upregulated by both hypoxia and ARSB
silencing in the hypoxia PCR array (Table 1), have AP-1 binding
sites, indicating a transcriptional mechanism by which they also
may be upregulated. The impact of changes in GAG sulfation on
nuclear translocation of oncogenes and other transcription factors
is a subject for further study which may have significant
implications for how genes are regulated by extra-nuclear, and
even extracellular, events.
In protists, yeasts, and plants, a sulfate assimilation pathway
(SAP) has been described by which sulfate is progressively reduced,
leading to production of glutathione, cysteine, and methionine
[29–36]. The SAP involves adenosine 59-phosphosulfate (APS) and
39-phosphoadenosine 59-phosphosulfate (PAPS). In mammalian
cells, PAPS synthetases (PAPSS1 and PAPSS2) have been
identified that combine the APS and PAPS functions and activate
sulfate [43,44]. The current study findings demonstrate that
diminished availability of sulfate due to reduced ARSB activity
following hypoxia in the human epithelial cells leads to decline in
the GSH/GSSG ratio and the sulfhydryl content, indicating a
profound impact on the overall cellular redox status. This suggests
the operation of a mechanism by which molecular oxygen can
regulate sulfate assimilation through activation of ARSB in
mammalian epithelial cells. The cascade of reactions from sulfate
to reduced sulfur in the sulfate assimilation pathway (SAP) (also
known as the sulfate activation complex) can be invoked to
integrate oxygen signaling, sulfate metabolism, and redox status,
since the cascade of reactions from sulfate to reduced sulfur would
be impaired due to reduced availability of sulfate following
inhibition of ARSB activity and reduced production of free sulfate.
The bacterial SAP involves interaction with several critical
enzymes, including thioredoxin and glutaredoxin that act as
hydrogen donors for PAPS reductase , and is thermodynam-
ically driven by GTP hydrolysis [35,36]
Effects on glutathione reduction impact on other complex redox
processes, such as those of monothiol glutaredoxins that utilize
reduced glutathione and a cysteine residue to bind to a bridging
[2Fe-2S] cluster . Decreased ratio of reduced glutathione to
glutathione disulfide is associated with selective promotion of
disulfide bond formation within cytoplasmic proteins, leading to
functional impairment . For normal cellular aerobic metab-
olism, coenzyme A (CoA), a sulfhydryl, is processed to the thioester
acetyl CoA. The production of acetyl-CoA from pyruvate by the
pyruvate dehydrogenase complex is inhibited by increase in the
ratio of acetyl-CoA to CoA, reflecting the impact of a thiol on
regulation of aerobic metabolism . Impairment of sulfate
assimilation, due to reduced activity of ARSB, in association with
reduced sulfhydryl content, would impair oxidative metabolism by
inhibition of pyruvate dehydrogenase. This suggests a mechanism
whereby impairment of aerobic metabolism, as hypothesized by
Warburg , might follow from decline in ARSB activity, and is
consistent with reported findings of reduced ARSB activity in
malignant cells [1,3,22,23].
The extensive, unexpected replication between the effects of
hypoxia and ARSB silencing in the BEC and NCM460 cells
suggests that ARSB mediates some intracellular effects of oxygen.
In turn, the effects of hypoxia on C4S and sulfated GAG content
indicate that a pathway of sulfate metabolism may be regulated by
oxygen through activation of ARSB. Effects of both hypoxia and
Figure 5. ARSB silencing and hypoxia reduce the GSH/GSSG
ratio. A. In the BEC, the ratio of reduced glutathione (GSH) to
glutathione disulfide declined significantly following hypoxia (10%
O264 h) and following ARSB silencing by siRNA (p,0.001). These
effects are consistent with decline in the overall reduced status of the
cells. B. Similarly, in the NCM460 cells, the reduced glutathione to
glutathione disulfide was markedly reduced, attributable to decline in
the reduced glutathione (p,0.001) following 10% O264 h. [BEC=
bronchial epithelial cell; ARSB=arylsulfatase B].
Arylsulfatase B and Oxygen Signaling
PLoS ONE | www.plosone.org7March 2012 | Volume 7 | Issue 3 | e33250
ARSB silencing on the reduced glutathione-glutathione disulfide
ratio and on the cellular sulfhydryl levels demonstrate ramifica-
tions of reduced availability of free sulfate on the overall cellular
redox state. Since removal of the 4-sulfate residue of N-
acetylgalactosamine-4-sulfate is the only known direct effect of
ARSB, ARSB may act as a redox switch that regulates the sulfate
assimilation pathway. Transcriptional effects may also be medi-
ated by ARSB through chondroitin-4-sulfation and the associated
changes in galectin binding to more or less highly sulfated GAGs.
By integrating the effect of oxygen with 1) chondroitin sulfation, 2)
sulfate assimilation and cellular redox status, and 3) transcriptional
regulation through variation in the interaction of galectins with
GAGs, ARSB is a critical link in our understanding of how
fundamental cell chemistry can regulate vital cell processes.
Materials and Methods
Culture of human bronchial and colonic epithelial cells
Normal human bronchial epithelial cells with retinoic acid
(NHBE; Lonza Walkersville, Inc., Walkersville MD) were grown
as recommended using Bronchial Epithelial Cell Growth Media
(BEGM; Lonza). NCM460 cells (INCELL, San Antonio, TX),
derived from normal human colonic mucosa, were grown as
recommended in M3:10A media (INCELL) . Cells were
initially grown in 12-well tissue culture clusters at 37uC, in a
humidified, 5% CO2, normoxic environment, and then were
placed in a modular incubator chamber (Billups-Rothenberg,
Inc., Del Mar, CA) which was loaded with a mixture of 85%
N2, 5% CO2, and 10% O2 for time periods ranging from
Figure 6. ARSB silencing and hypoxia reduce the total cellular sulfhydryl content. A. Overall decline in the total cellular sulfhydryl content
in the BEC followed both hypoxia (10% O264 h) and ARSB silencing, consistent with the observed effect on the reduced glutathione and the overall
change in the cellular redox state (p,0.001). Total sulfhydryl content is composed of inorganic and protein thiols, and the inorganic thiol did not
change significantly, indicating that the change in total thiols was attributable to the protein-associated thiols. B. Similar decline in the thiol cellular
sulfhydryl content occurred in the NCM460 cells following either hypoxia (10% O264 h) or ARSB silencing (p,0.001). The inorganic thiol content did
not change significantly, indicating that the change occurred in the protein thiol component. [BEC=bronchial epithelial cell; ARSB=arylsulfatase B].
Arylsulfatase B and Oxygen Signaling
PLoS ONE | www.plosone.org8March 2012 | Volume 7 | Issue 3 | e33250
15 minutes to 24 hours. The sealed chamber was placed in a
humidified incubator at 37uC. For experiments to determine the
recovery from hypoxia, cell preparations were returned to the
original normoxic conditions for 4 hours, following 4 hours of
Sulfatase activity assays
Determinations of ARSB activity were performed using a
fluorimetric assay with the substrate 4-methylumbilliferyl sulfate
(4-MUS) [22–24]. 20 ml of cell homogenate or of spent media and
80 ml of assay buffer (0.05 M Na acetate buffer, pH 5.6) were
combined with 100 ml of substrate (5 mM 4-MUS in assay buffer)
in wells of a microplate. The microplate was incubated for
30 minutes at 37uC. The reaction was stopped by 150 ml of stop
buffer (Glycine-Carbonate buffer, pH 10.7), and fluorescence was
measured at 360 nm (excitation) and 465 nm (emission) in a
microplate reader) (FLUOstar, BMG LABTECH, Inc., Cary,
NC). Activity of galactose-6-sulfatase (GALNS), steroid sulfatase
(STS), and arylsulfatase A (ARSA) was also measured in the
epithelial cells, as previously described [22–24].
ARSB silencing and overexpression
Cell preparations that were subjected to ARSB silencing or
overexpression were grown in 12-well clusters. At 60% confluency,
cells were transfected with siRNA for ARSB (Qiagen, Valencia,
CA) or by control siRNA for 24 hours, as previously detailed
[3,4,6]. Overexpression was performed with an ARSB construct
(OriGene Technologies, Inc., Rockville, MD) as previously
Induction of hypoxia
Measurements of pO2 in the cell cultures were performed using
an oxygen needle probe (Lazar Research Laboratories, Los
Angeles, CA), following exposure to either the normoxic or
hypoxic environment at 1 and 4 hours. Confirmation of hypoxic
conditions was achieved by these measurements, which indicated
decline of ,40% in the oxygen saturation of the spent media
under hypoxic conditions vs. the normoxic control. Selection of
10% O2environment was based on conditions in previous reports
indicating that a 50% reduction in the available oxygen produced
significant changes, but without severe toxicity [50,51].
Figure 7. Correlation between Ct values of hypoxia and ARSB silencing for 84 hypoxia-associated genes and 5 housekeeping genes
in NCM460 cells. The average corrected Ct values for the hypoxic (10% O264 h) cells and the ARSB silenced cells had a correlation coefficient
r=0.994. The direction and extent of change in mRNA expression was similar for each of the genes, using simultaneous determinations performed in
triplicate on the PCR arrays.
Table 1. Genes with expression significantly increased by both hypoxia and ARSB silencing.
P-valueFold change P-valueFold change
COL1A1Collagen, type I, alpha 1 0.0311.7 0.00462.0
HMOX1Heme oxygenase (decycling) 10.030 1.60.052 1.7
IL6 Interleukin 6 (interferon, beta-20.0023 9.3 0.00475.5
PLAUPlasminogen activator, urokinase0.016 2.00.0040 2.5
PLOD3 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 30.022 1.6 0.00972.2
PTX3Pentraxin-related gene, rapidly induced by IL-1 beta0.013 3.3 0.0030 5.2
Arylsulfatase B and Oxygen Signaling
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SUMF-1 assay and SUMF-1 silencing by siRNA
Sulfatase modifying factor (SUMF)-1 in the control and treated
cell extracts was detected by ELISA (USCN Life Sciences, Inc.,
Wuhan, China) that captured cellular SUMF-1 in microtiter wells
coated with anti-SUMF-1 monoclonal antibody. SUMF-1 was
detected by biotinylated SUMF-1 polyclonal antibody and avidin-
horseradish peroxidase (HRP). The enzyme activity of bound
HRP was determined by adding hydrogen peroxide-tetramethyl-
benzidine (TMB) chromogenic substrate. The magnitude of the
optical density of the developed color was measured in an ELISA
plate reader at 450 nm. SUMF-1 concentrations were determined
from a standard curve made with known concentrations of SUMF-
1. Sample values were normalized with the total protein
concentrations determined by BCA protein assay kit (Pierce,
Thermo Fisher Scientific, Rockford, IL). SUMF-1 ELISA was
used to detect the extent of silencing following treatment of cells by
commercial SUMF-1 siRNA (Qiagen). Four different siRNAs
were tested for their effect on SUMF1 expression, and the siRNA
prepared against the target sequence 59-GTCGAGGAGGCCTG-
CATAATA-39 was selected for knockdown of SUMF-1 in the
cells, following an established protocol for gene silencing [3,4].
Measurements of sulfated GAG and C4S levels
Measurements of total sulfated GAG and C4S were performed
as previously described . Briefly, the BlyscanTMassay kit
(Biocolor Ltd, Newtownabbey, N. Ireland) was used for detection
of the sulfated GAG, based on the reaction of 1,9-dimethylmethy-
lene blue with the sulfated oligosaccharides in the GAG chains.
C4S content was determined following immunoprecipitation with
C4S antibody, as reported previously .
Activated HIF-1a ELISA
Nuclear extracts of treated and control cells were prepared
using a nuclear extract kit [Active Motif, Carlsbad, CA]. Activated
HIF-1a in the nuclear extract of the control and treated cells was
determined by a commercial oligonucleotide-based ELISA (R & D
Systems, MN). Activated HIF-1a in the nuclear extract bound
to a biotinylated double-stranded oligonucleotide containing a
Figure 8. Galectin-3 co-immunoprecipitated with C4S declined
following hypoxia and ARSB silencing, and nuclear galectin-3
increased. A, B. The galectin-3 that co-immunoprecipitated with C4S
in the BEC declined significantly following both hypoxia (10% O264 h)
and ARSB silencing (p,0.001), consistent with reduced association of
galectin-3 with more highly sulfated C4S. C, D. Galectin-3 content in
the nuclear fraction of the BEC increased significantly following both
ARSB silencing and hypoxia (10% O264 h) (p,0.001). [BEC=bronchial
epithelial cell; ARSB=arylsulfatase B].
Figure 9. Activated AP-1 increased following ARSB silencing or
hypoxia in the BEC. A. Nuclear c-Jun significantly increased following
ARSB silencing or 10% O264 h. B. Similarly, c-Fos significantly increased
following ARSB silencing or 10% O264 h. [BEC=bronchial epithelial cell;
Arylsulfatase B and Oxygen Signaling
PLoS ONE | www.plosone.org10 March 2012 | Volume 7 | Issue 3 | e33250
consensus sequence of the HIF-1a binding site, and the HIF-1a-
oligonucleotide complexes were captured by an immobilized
antibody specific for HIF-1a that was coated onto the wells of a
microtiter plate. Captured HIF-1a molecules were subsequently
detected by streptavidin-horseradish peroxidase (HRP), and HRP
activity was determined by adding hydrogen peroxide-tetra-
methylbenzidine (TMB) chromogenic substrate. The optical
density for the developed color was measured in an ELISA reader
(FLUOstar, BMG) at 450 nm, after stopping the reaction with 2N
sulfuric acid. The intensity of the developed color was propor-
tionate to the quantity of activated HIF-1a in each sample. The
sample values were normalized with the total cell protein and
expressed as a percentage of control.
PCR array for hypoxia-associated gene expression
Hypoxic PCR gene array (SABiosciences, Frederick, MD) to
detect changes in expression of 84 hypoxia-associated genes was
performed in triplicate using 384-well microplates, in which 4-
samples were tested simultaneously for each of the mRNAs of
interest. Samples included: NCM460 cells in which ARSB was
silenced by siRNA, NCM460 cells treated by control siRNA,
untreated NCM460 cells, and NCM460 cells exposed to 10%
oxygen for 4 hours. Plates were analyzed by calculations of cycle
threshold (Ct) values for each of the wells, and by normalization
using the Ct values to controls from five housekeeping genes.
Comparisons were made between the mean Ct values (n of 3) for
each of the four conditions using the normalized Ct values.
Statistical significance was determined by t-test that compared the
normalized Ct values for each of the genes of interest. Correlation
coefficient (r) was calculated using Excel software.
QPCR was performed for HIF-1a with forward primer 59-
GCTATTTGCGTGTGAGGAAAC-39 and reverse primer 59-
CACCATCATCTGTGAGAACCA-39. QPCR was performed
using established methods .
Glutathione (GSH/GSSG/Total) assay
GSH, GSSG, and total glutathione in the control and treated
cells were determined (Glutathione Detection Kit, BioVision, Inc.,
Mountain View, CA). Samples were collected on 6N perchloric
acid on ice to avoid oxidation of labile GSH. GSH was determined
by reaction of cell samples with O-phthalaldehyde (OPA). OPA
reacted with cellular GSH, but not GSSG, to generate
fluorescence. GSH+GSSH total was determined by first adding
a reducing agent which, converted GSSG to GSH, and then
reacting with OPA to produce fluorescence. To measure GSSG
specifically, a GSH quencher was added initially to remove GSH,
preventing the reaction with OPA, but not interacting with GSSG.
Reducing agent was then added to destroy excess quencher and to
convert GSSG to GSH. The fluorescence was measured in a
fluorescence plate reader (FLUOstar) with excitation and emission
wavelengths of 340 nm and 420 nm, respectively.
Total, inorganic, and protein sulfhydryl determinations
Cellular sulfhydryl in the control and treated samples were
determined by a commercial assay (Molecular Probes, Eugene,
OR) [52,53]. Inorganic thiols in the samples reduced a disulfide
bond, releasing the active enzyme papain from its inactive S-S
form. The activity of the enzyme was then measured using the
chromogenic papain substrate, N-benzoyl-L-arginine, p-nitroani-
lide (L-BAPNA). The intensity of the developed color was
proportionate to the quantity of thiols in each sample. To measure
the total (inorganic and protein-associated) thiols, cystamine was
added to the reaction, which permitted the detection of poorly
accessible thiols on proteins with high pKa values. Cystamine, a
disulfide, underwent an exchange reaction with protein thiols,
yielding 2-mercaptoethylamine (cysteamine), which then released
active papain. Active papain then acted on L-BAPNA to develop
color, and absorbance was measured in a spectrophotometer at
410 nm. The protein sulfhydryl component was calculated as the
difference between the total and the inorganic components.
Figure 10. Schematic illustration of oxygenRARSBRC4SRgalectin-3RAP-1RHIF-1a signaling pathway. Normoxic and hypoxic
conditions lead to differences in signaling, attributable to reduced ARSB activity leading to increase in chondroitin-4-sulfation, reduced galectin-3
binding to the more highly sulfated C4S, increased nuclear translocalization of galectin-3, and increased transcriptional events in association with
activation of AP-1, including increased expression of HIF-1a. [ARSB=arylsulfatase B; FGE=formylglycine (oxoalanine) generating enzyme;
Arylsulfatase B and Oxygen Signaling
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Galectin-3 measurements by ELISA in cell fractions and
following immunoprecipitation with chondroitin-4-
Galectin-3 was determined by a sandwich ELISA kit (R&D
Systems, Minneapolis, MN) for human galectin-3. The wells of a
microtiter plate were coated with specific anti-galectin-3 mono-
clonal antibody, and nonspecific sites were blocked by a blocking
buffer with 1% bovine serum albumin (BSA). Cell fractionation
was performed, separating the cytoplasmic and nuclear fractions
by centrifugation for 30 seconds at 14,000 g. The galectin-3 from
the spent media, cytoplasmic and nuclear fractions were separately
captured into the microtiter wells by specific galectin-3 antibody.
Captured Galectin-3 was then detected by biotin-conjugated
secondary galectin-3 antibody and streptavidin-horseradish per-
oxidase (HRP). Hydrogen peroxide-tetramethylbenzidine (TMB)
chromogenic substrate was used to develop the color, and the
intensity of color was measured at 450 nm in an ELISA plate
reader (FLUOstar, BMG). The galectin-3 concentrations were
extrapolated from a standard curve, and sample values were
normalized with total protein content (BCA Protein Assay Kit,
Pierce (Rockford, IL). In addition, galectin-3 was measured
following immunoprecipitation of the BEC cells with C4S
antibody (4D1; Abnova, Novus Biologicals, Littleton, CO). Cell
lysates were prepared from treated and control cells. C4S (4D1)
antibody binds to native C4S and cross-reactivity with other
chondroitin sulfates is not significant . The antibody was
added to the cell lysates (1 mg/mg of protein), and tubes were
rotated overnight in a shaker at 4uC. Next, 100 ml of pre-washed
Protein L-agarose (Santa Cruz Biotechnology) was added to each
tube, and the tubes were incubated overnight at 4uC. The Protein
L-agarose-treated beads were washed three times with phosphate-
buffered saline containing Protease Inhibitor Mixture. The
precipitate was eluted with dye-free elution buffer and subjected
to galectin-3 assay as described above.
Detection of nuclear AP-1 by oligonucleotide-based
Oligonucleotide binding assay (Active Motif, Carlsbad, CA) was
used to detect nuclear c-Jun and c-Fos in the BEC following
hypoxia (10% oxygen64 hr) or ARSB silencing by siRNA.
Nuclear extracts from treated and control cells were prepared
using a nuclear extract preparation kit (Active Motif). Activated
AP-1 components in the samples were detected by oligonucleotide-
based ELISA. The nuclear extracts were added to the wells of the
96-well microtiter plate, pre-coated with an AP-1 consensus
oligonucleotide sequence (59-TGAGTCA-39). The AP-1 proteins
from the nuclear extract attached to the coated oligonucleotides,
and the bound c-Jun and c-Fos were detected by specific
antibodies and anti-rabbit-HRP-conjugated IgG. The specificity
of the binding of c-Jun and c-Fos with the coated nucleotide
sequence was determined by comparison with the binding when a
known quantity of free consensus nucleotide or mutated nucleotide
was added in the reaction buffer. Colorimetric readout was
performed with hydrogen peroxide-TMB chromogenic substrate.
After the reaction was stopped with 2N sulfuric acid, the optical
density of the developed color was measured in an ELISA plate
reader (FLUOstar, BMG) at 450 nm. The intensity of the
developed color proportionately represents the quantity of c-Jun
or c-Fos in each sample. The sample values were normalized with
the total cell protein and expressed as percent of untreated control.
Experiments were performed with at least three independent
biological samples and with technical replicates of all measure-
ments. P-values #0.05 were considered statistically significant.
One-way ANOVA with Tukey-Kramer post-test was performed
for analysis of the significance of differences between measure-
ments, unless stated otherwise. For the PCR array, statistical
significance was corrected for multiple comparisons, and Ct values
were adjusted by normalization with five housekeeping genes.
of hypoxia-associated genes in PCR array following
hypoxia or ARSB silencing.
Corrected average cycle threshold (Ct) values
The authors acknowledge the contributions of Kumar Kotlo, Ph.D. to
overexpression experiments and of Robert Danziger, M.D. to study design.
Conceived and designed the experiments: JKT SB. Performed the
experiments: SB. Analyzed the data: SB JKT. Contributed reagents/
materials/analysis tools: SB JKT. Wrote the paper: JKT.
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Arylsulfatase B and Oxygen Signaling
PLoS ONE | www.plosone.org13March 2012 | Volume 7 | Issue 3 | e33250