Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle.
ABSTRACT The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.
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ABSTRACT: Mitochondrial density in skeletal muscle fibers is governed by the demand for aerobic ATP production, but the heterogeneous distribution of these mitochondria appears to be governed by constraints associated with oxygen diffusion. We propose that each muscle fiber has an optimal mitochondrial distribution at which it attains a near maximal rate of ATP consumption (RATPase) while mitochondria are exposed to a minimal oxygen concentration, thus minimizing reactive oxygen species (ROS) production. We developed a coupled reaction-diffusion/cellular automata (CA) mathematical model of mitochondrial function and considered 4 fiber types in mouse extensor digitorum longus (EDL) and soleus (SOL) muscle. The developed mathematical model uses a reaction - diffusion analysis of metabolites including oxygen, ATP, ADP, phosphate and phosphocreatine (PCr) involved in energy metabolism and mitochondrial function. A CA approach governing mitochondrial life cycles in response to the metabolic state of the fiber was superimposed and coupled to the reaction-diffusion approach. The model results show the sensitivity of important model outputs such as the RATPase, effectiveness factor (η) and average oxygen concentration available at each mitochondrion to local oxygen concentration in the fibers through variation in the CA model parameter θdet, which defines the sensitivity of mitochondrial death to the oxygen concentration. The predicted optimal mitochondrial distributions matched previous experimental findings. Deviations from this optimal distribution corresponding to higher CA model parameter values (a more uniform mitochondrial distribution) lead to lower aerobic rates. In contrast, distributions corresponding to lower CA model parameter values (a more asymmetric distribution) lead to an increased exposure of mitochondria to oxygen, usually without substantial increases in aerobic rates, which would presumably result in increased ROS production and thus increased risks of cytotoxicity. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.Biotechnology and Bioengineering 04/2013; · 4.16 Impact Factor
- Comparative Biochemistry and Physiology D. 4:121-127.
Hyperoxia-mediated oxidative stress increases expression of
UCP3 mRNA and protein in skeletal muscle
Pierre Flandina,*, Yves Donatib, Constance Barazzone-Argiroffob, Patrick Muzzina
aDepartment of Cell Physiology and Metabolism, Centre Me ´dical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland
bDepartment of Pathology, Centre Me ´dical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland
Received 15 March 2005; revised 20 April 2005; accepted 26 April 2005
Available online 23 May 2005
Edited by Vladimir Skulachev
protein expressed mainly in skeletal muscle. Among several
hypotheses for its physiological function, UCP3 has been pro-
posed to prevent excessive production of reactive oxygen species.
In the present study, we evaluated the effect of an oxidative stress
induced by hyperoxia on UCP3 expression in mouse skeletal
muscle and C2C12 myotubes. We found that the hyperoxia-med-
iated oxidative stress was associated with a 5-fold and 3-fold in-
crease of UCP3 mRNA and protein levels, respectively, in mouse
muscle. Hyperoxia also enhanced reactive oxygen species pro-
duction and UCP3 mRNA expression in C2C12 myotubes.
Our findings support the view that both in vivo and in vitro
UCP3 may modulate reactive oxygen species production in re-
sponse to an oxidative stress.
? ? 2005 Federation of European Biochemical Societies. Published
by Elsevier B.V. All rights reserved.
The uncoupling protein-3 (UCP3) is a mitochondrial
Keywords: Uncoupling protein; UCP3; Hyperoxia; Oxidative
stress; Reactive oxygen species; Antioxidant enzyme
The uncoupling protein-3 (UCP3) belongs to the mitochon-
drial carrier family. This protein is mainly expressed in skeletal
muscle and in brown adipose tissue of rodents. It has also been
shown to be present in the skeletal muscle of other species,
including human  and chicken . Despite intensive work
since its identification in 1997 by our group and other, its phys-
iological function remains still unknown. UCP3 was originally
proposed to uncouple the oxidative phosphorylation by dissi-
pating the mitochondrial proton gradient, but its uncoupling
activity is still debated. An overall view of studies on UCP3
function to date suggests a role for this protein in the regulation
of energy metabolism, energy partitioning, mitochondrial pro-
duction of reactive oxygen species (ROS) and in the detoxifica-
tion of oxidant molecules by exporting peroxidized fatty acid
out of the mitochondrial matrix [3–7]. ROS regroup molecules
such as superoxide anion, hydroxyl radical and hydrogen per-
oxide and possess strong oxidative capacity. The potential role
forUCP3 inmodulatingROS formationissupported byin vivo
and in vitro studies. For instance, mice lacking UCP3 exhibited
an increase in ROS level in their muscles , and UCP3 overex-
pression in primary culture of neurons resulted in an inhibition
of hyperglycemia-induced oxidative stress . ROS, at their
in isolated mitochondria the GDP sensitive-uncoupling activity
of UCP3 depends on the presence of ROS .
Production of ROS during exposure to hyperoxia is widely
held to be responsible for the lung injury seen in oxygen-
exposed animals.Hyperoxia hasalso beenusedas anexper-
imental procedureto induceoxidativestress in otherorgansuch
as heart and skeletal muscle [12,13]. For instance, it has been
shown that, in aged rats, exposure to high concentration of
oxygen for 60 h resulted in an increased antioxidant activity
of catalase and superoxide dismutase in skeletal muscle .
As mentioned above, it has been reported that manipulating
UCP3 gene expression influenced ROS production. In the
present study, we used a complementary approach to deter-
mine the relationship between UCP3 and ROS. We investi-
gated the effect of hyperoxia-induced oxidative stress on
muscle UCP3 expression in both in vivo and in vitro models.
To determine the in vivo impact of hyperoxia on UCP3 expres-
sion, we exposed C57BL/6 mice in 100% oxygen for 72 h, as-
sessed markers of oxidative stress and measured UCP3
mRNA and protein levels. In parallel, we examined whether
in vitro hyperoxia induced ROS formation and regulated
UCP3 expression in the murine skeletal muscle C2C12 cell line.
Our results show that a well-defined condition of oxidative
stress, namely hyperoxia, induced an increase in UCP3 expres-
sion both in mouse skeletal muscle and C2C12 myotubes, sug-
gesting that this protein might participate to ROS metabolism.
2. Materials and methods
2.1. Exposure of mice to hyperoxia
Two- to three-month old C57BL/6 male mice (Charles River,
France) were exposed to room air or hyperoxic conditions by delivery
of 100% oxygen to a sealed Plexiglas chamber for 72 h as previously
described . The mice were given free access to food and water
and were exposed to a 12-h light–dark cycle. At the end of oxygen
exposure, mice were killed and skeletal muscles were rapidly dissected,
Abbreviations: CM-H2DCFDA, 5-(and-6)-chloromethyl-20,70-dichlo-
rodihydrofluorescein diacetate, acetyl ester; CuZn-SOD, copper/zinc-
superoxide dismutase; DMEM, Dulbecco?s modified Eagle?s medium;
HBSS, Hank?s balanced salt solutions; PCR, polymerase chain reac-
tion; ROS, reactive oxygen species; UCP2, uncoupling protein-2;
UCP3, uncoupling protein-3
E-mail addresses: firstname.lastname@example.org (P. Flandin),
email@example.com (P. Muzzin).
0014-5793/$30.00 ? 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
FEBS 29629 FEBS Letters 579 (2005) 3411–3415
frozen in liquid nitrogen and stored at ?80 ?C until further analysis.
All procedures used were approved by the Office Ve ´te ´rinaire Cantonal
of Geneva, Switzerland.
2.2. Exposure of cells to hyperoxia
C2C12 myoblast cells were grown in Dulbecco?s modified Eagle?s
medium (DMEM) high glucose (4.5 g/L) supplemented with 10% fetal
bovine serum until they reach 90% confluence. In order to differentiate
(1 mg/L) DMEM supplemented with 2% horse serum. After 5 addi-
tional days of culture, a large proportion of long multinucleated myotu-
bes was observed among the myoblasts. These differentiated cells were
placed under 95% oxygen/5% CO2(hyperoxia) or maintained in normal
air condition (normoxia) for 48 h. After exposure, cells were used either
for measurement of ROS production or RNA extraction (see below).
2.3. Measurement of ROS production
ROS were detected using fluorescence assay on C2C12 cells plated in
96 microwell optical bottom plates (Nunc GmBH&Co. KG, Wiesba-
den Germany). Myotubes under hyperoxia or normoxia were quickly
washed in Hank?s balanced salt solutions (HBSS) and incubated with
10 lM of5-(and-6)-chloromethyl-20,70-dichlorodihydrofluorescein
diacetate, acetyl ester (CM-H2DCFDA) membrane-permeable dye
(Molecular Probes, Europe BV, Leiden, The Netherland) at 37 ?C
for 10 min. Then, cells were washed twice with HBSS to remove excess
dye. After addition of HBSS, cellular ROS accumulation was deter-
mined by recording the fluorescence (excitation wavelength: 485 nm
and emission wavelength: 535 nm) for 10 min using a thermostate con-
trolled (37 ?C) microplate reader.
2.4. Northern blotting
Total RNA from mouse skeletal muscle was isolated by the method
of Chomczynski and Sacchi . Northern blot analysis was performed
as described previously . Full length cDNAs were used as probes for
the detection of UCP3, CuZn-superoxide dismutase (CuZn-SOD) and
catalase mRNAs. Human CuZn-SOD and human catalase cDNAs
were kindly provided by the Dr. Lan Jornot. Human b-actin probe
was used to ensure that equivalent RNA amounts were blotted on
the membrane. The signals were quantified by scanning photodensi-
tometry and normalized using the corresponding actin mRNA values.
2.5. Real-time PCR
The determination of UCP3 mRNA levels in myotubes was per-
formed by quantitative polymerase chain reaction (PCR). Total RNA
was prepared using the NucleoSpin RNA II kit mRNA (Macherey-Na-
gel, Du ¨ren, Germany) according to the manufacturer?s instructions.
Oligo-dT first strand cDNA were synthesized using the Superscripte
II RNase H Reverse Transcription kit (Invitrogene, Life technologies,
Basel, Switzerland) according to the manufacturer?s instructions. Real-
time PCR was performed using ABI rapid thermal cycler system and a
SYBR Green PCR master mix according to the manufacturer?s instruc-
tions. Cyclophilin A was used as a control to account for any variations
due to the efficiencies of the reverse transcription and PCR. UCP3
oligonucleotide primers used were: upstream 50-GGAGTCTCAC-
CTGTTTACTGACAACT-30and downstream 50-GCACAGAAGC-
CAGCTCCAA-30(GenBank Accession No. NM009464). Cyclophilin
A oligonucleotide primers used were: upstream 50-AGCACTGGGGA-
GAAAGGATT-30and downstream 50-CATGCCTTCTTTCACCT-
TCC-30(GenBank Accession No XM355936). The conditions of
PCR were a step at 50 ?C for 2 min followed by a denaturing step at
95 ?C for 10 min and by 50 cycles at 95 ?C for 15 s and 60 ?C for
1 min. After each run, a relative quantification of amplified PCR prod-
ucts in the different samples was performed. For this purpose, standard
curves were constructed for the gene of interest as well as for cyclophi-
lin. The results are expressed as the ratio between the concentration of
the target gene and that of cyclophilin A.
2.6. Mitochondria preparation
Mitochondria of skeletal muscle were prepared as previously de-
scribed . Mitochondrial protein concentrations were determined
according to Bradford et al. , using the Bio-Rad Protein Assay
(Bio-Rad Laboratories, Hercules, CA, USA), with bovine serum albu-
min as a standard.
2.7. Western blotting
Muscle mitochondria were prepared and Western blot performed as
previously described , using a rabbit polyclonal primary antibody
raised against human UCP3 protein (CabrX; Research Diagnostics
Inc., San Antonio, LA, USA). Membranes were subsequently reblot-
ted with a monoclonal antibody specific for prohibitin (Santa Cruz)
to ensure that equivalent amount of mitochondria proteins was loaded
onto the gel. The signals were detected by chemiluminescence using a
standard ECL kit, and developed on a Hyperfilm ECL film. They were
quantified by scanning photodensitometry of the autoradiograms
using ImageQuant Software version 3.3 of Molecular Dynamics (Sun-
2.8. Aconitase activity
To measure aconitase activity, we used the method described by
Hausladen and Fridovich  with some modifications. Briefly,
200 lg of mitochondrial proteins from anterior leg muscles were dis-
rupted by 3 times frozen–thaw in aconitase buffer containing Tris–
HCl 50 mM, pH 7.4, sodium citrate 5 mM, cysteine 1 mM and MnCl2
0.6 mM. Mitochondrial extracts were then added to an aconitase buffer
containing 10 units of NADP+-dependent isocitrate dehydrogenase.
The reaction started by adding NADP+0.2 mM. Basal NADPH
absorbance was measured every minute in a spectrophotometer and
when stable values were obtained NADP+was injected. Calculation
of aconitase activity was based on quantity of NADPH produced in
the first 10 min after injection of NADP+. Aconitase activity was ex-
pressed as nmoles NADPH produced per mg of mitochondrial protein
per minute. To convert OD340in nmoles, we used a standard curve
with 0.5, 5, 25 and 50 nmol of NADPH.
2.9. Statistical analysis
Data are expressed as means ± S.E.M. Significance was evaluated
using the unpaired Student?s t test. A P value less than 0.05 was con-
sidered statistically significant.
3.1. Hyperoxia induces an oxidative stress in skeletal muscle of
We first assessed the oxidative stress level in skeletal muscle
of mice exposed to hyperoxia for 72 h (hyperoxia) or main-
tained in normoxic conditions (normoxia). As an in vivo indi-
cator of mitochondrial ROS accumulation we determined the
aconitase activity in muscle mitochondria. Loss in aconitase
activity is interpreted as a measure of oxidative stress. As
shown in Fig. 1A, aconitase activity was significantly decreased
by 38% in hyperoxic mice compared with normoxic controls.
The maximal aconitase activities were similar between norm-
oxic and hyperoxic mitochondria. Then, we determined the
mRNA levels of CuZn-SOD, catalase and b-actin in skeletal
muscle. Fig. 1B show that hyperoxia induced a significant
3.0-fold and 2.6-fold increase in mRNA levels of CuZn-SOD
and catalase, respectively. b-Actin mRNA level was found to
be unchanged by hyperoxia. These data indicate that exposure
to 100% oxygen for 72 h causes an oxidative stress in the
mouse skeletal muscle.
3.2. UCP3 mRNA and protein are increased in skeletal muscle
of hyperoxic mice
To examine the effect of oxidative stress on UCP3 expres-
sion, we compared the UCP3 mRNA level in skeletal muscle
of hyperoxic mice with those of normoxic controls. As shown
in Fig. 2A, oxygen exposure produced a 5-fold increase in
UCP3 mRNA. Then, we determined the UCP3 and prohibitin
protein levels in muscle mitochondria. Fig. 2B shows that
UCP3 protein expression was also upregulated in muscle
P. Flandin et al. / FEBS Letters 579 (2005) 3411–3415
mitochondria of hyperoxic mice. No difference was observed in
the level of prohibitin between normoxia and hyperoxia condi-
tions. These results show that 72 h hyperoxia induced a
marked increase of UCP3 mRNA and protein expression in
mouse skeletal muscle.
3.3. 48 h hyperoxia induces UCP3 mRNA expression and
oxidative stress in C2C12 myotubes
To examine the direct effect of hyperoxia on UCP3 expres-
sion in muscle cells, we performed in vitro experiment on con-
fluent C2C12 myotubes exposed to either normoxic (control)
or hyperoxic conditions for 48 h. The UCP3 mRNA expres-
sion relative to that cyclophilin, which is considered to be a
reference housekeeping gene, was significantly increased by
1.9-fold in hyperoxic myotubes compared to control cells
(Fig. 3A). We also evaluated the level of oxidative stress in
C2C12 myotubes after 48 h exposure to hyperoxia. Using a
dichlorofluorescein probe (CM-H2DCFDA) to detect ROS,
we found a 40% increase in oxidative stress level of hyperoxic
myotubes compared to controls (Fig. 3B). Thus, in C2C12
myotubes, hyperoxia generates an augmentation of ROS pro-
duction associated to an increase in UCP3 mRNA expression,
suggesting that UCP3 expression could be control by the level
of cellular oxidative stress.
Here, we report that hyperoxia induced an increase in UCP3
mRNA and protein expression in skeletal muscle and in UCP3
mRNA level in C2C12 myotubes. In parallel to the increase in
UCP3 expression by hyperoxia, we also observed an increase
in the level of oxidative stress. Our data suggests that the oxi-
dative stress produced by hyperoxia could be the stimulator of
Although exposure to 100% oxygen is well known to induce a
marked oxidative stress and oxidative cell damages in lung, the
effect of hyperoxia on oxidative stress levels in skeletal muscle
has been rarely studied . We therefore determined the level
of oxidative stress generated in skeletal muscle of hyperoxic
mice by two indirect but commonly used methods; the measure-
ments of aconitase activity and antioxidant enzymes mRNA
expression. Aconitase is an enzyme in the tricarboxylic acid cy-
cle that is inhibited by superoxide radicals. Loss of aconitase
activity is a widely used index of oxidative stress. We observed
that the relative aconitase activity (% of the maximal activity) in
hyperoxia represents 60% of the activity in normoxic condition.
In line with our observation, similar decrease in aconitase activ-
ity was reported in mitochondria of superoxide dismutase-2
heterozygous knockout mice . We also observed an increase
in the gene expression of CuZn-SOD and catalase mRNA after
72 h hyperoxia suggesting an increase in ROS level. Hyperoxia
seems to induce ROS production in mouse skeletal muscle in a
time-dependent manner as 24 h hyperoxia exposure did not af-
fect catalase and CuZn-SOD mRNA expression (data not
shown). UCP3 mRNA and protein levels were also unchanged
in 24 h hyperoxia compared to normoxia (data not shown).
The observation that 72 h hyperoxia induced gene expres-
sion of CuZn-SOD and catalase, suggest that some ROS-
dependent signaling pathways have been turn on. This is in
contrast with the observation of Amicarelli et al , who re-
ported that 60 h hyperoxia did not result in significant changes
of antioxidant enzymes activities in muscle homogenate of
young rats. It is possible that the difference between their re-
sults and ours might be due to the use of two different animal
models, i.e., exposure to oxygen for 60 h in rat vs. 72 h in
mouse. However, the fact that there is not a systematic corre-
lation between activities of antioxidant enzymes and their
mRNA expression make difficult the comparison between the
two studies. To support data obtained in hyperoxic muscle,
we demonstrated by measuring ROS production that hyper-
oxia generated an oxidative stress in cultured myotubes. Taken
together, our data indicate that 72 h hyperoxia is a condition
that creates production of free radicals in skeletal muscle.
Hyperoxia is known to turn on several ROS-dependent sig-
naling pathways such as MAPK, NF-jB or AP-1 pathways in
endothelial and epithelial cells . It is unknown whether
UCP3 expression could be directly induced by one or many
of these pathways. However it can be mentioned that TNF-
a, which induces mitochondrial ROS production and activates
NF-jB signaling in C2C12 myotubes , upregulates UCP3
mRNA expression in rat skeletal muscle . Based on these
reports, one may hypothesize that the NF-jB pathway
Fig. 1. Oxidative stress evaluation in skeletal muscle of hyperoxic mice.
(A) Aconitase activity in mouse muscle of normoxic and hyperoxic
mice. Aconitase activity was measured as described under Section 2.
Results are expressed as the percent of the maximal aconitase activity.
Values are means ± S.E.M.; n = 10. (B) Catalase and CuZn-SOD
mRNA levels in normoxic and hyperoxic mice. The expressions of
catalase and superoxide dismutase are shown relative to that of b-actin.
The ratio of the normoxic values is considered as 1.0. Results are
expressed as means ± S.E.M. of arbitrary units; n = 5. (C) Represen-
tative catalase, CuZn-SOD and actin mRNA signals (Norm. =
normoxia and Hyp. = hyperoxia).\, P < 0.05;\\\, P < 0.0005, unpaired
Student?s t test.
P. Flandin et al. / FEBS Letters 579 (2005) 3411–3415
mediates the effect of ROS on UCP3 gene expression. Future
studies will address this hypothesis.
The relationship between UCP3 and ROS has mostly been
studied by quantifying the degree of ROS production in mod-
els lacking or overexpressing UCP3 [8,25]. Here, we deter-
minedtheregulation of UCP3
expression in response to an oxidative stress. We showed that
hyperoxia increased UCP3 expression in mouse skeletal muscle
and in C2C12 myotubes. As 72 h of hyperoxia significantly in-
duced an oxidative stress in muscle cells both in vivo and in vi-
tro, we might hypothesize that the induction of UCP3
expression would result from an enhanced ROS production.
Recent studies, which have investigated the expression of
uncoupling proteins in relation to oxidative stress, showed that
the oxidative stress is an inducer of uncoupling protein-2 and -
5 [26–28]. For instance, the increased production of mitochon-
drial ROS is associated with an increase in the mitochondrial
content of UCP2 in hepatocytes . Two hours exposure to
hydrogen peroxide increased by 1.5-fold UCP2 mRNA expres-
sion in INS-1 cells . In the present study, we showed that an
oxidative stress can also increase the expression of another
member of the uncoupling protein family that is UCP3, both
in vivo, in mouse skeletal muscle, and in vitro, in C2C12 myo-
tubes. The possibility that UCP3 may act as a regulator of
mitochondrial free radical generation or an exporter of perox-
idized fatty acids in muscle mitochondria remains to be inves-
tigated. It could be interesting in further study to test whether
exposition to hyperoxia may induce an increase in the proton
leak on isolated mitochondria or cells.
Acknowledgments: This work was supported by Grant No. 31-
54306.98/2 from the Swiss National Science Foundation and a grant-
in-aid of the Helmut Horten Foundation. We thank Mrs. Francoise
Kuhne for her excellent technical assistance and Dr. Lan Jornot for ad-
vice and helpful discussion.
 Boss, O., Samec, S., Paoloni-Giacobino, A., Rossier, C., Dulloo,
A., Seydoux, J., Muzzin, P. and Giacobino, J.P. (1997)
Fig. 2. UCP3 mRNA and protein expression in skeletal muscle of hyperoxic mice. (A) UCP3 mRNA levels in mouse muscle of normoxic and
hyperoxic mice. The expression of UCP3 is shown relative to that of b-actin. The ratio of the control values is considered as 1.0. Results are expressed
as means ± S.E.M. of arbitrary units; n = 5. (B) UCP3 protein levels in muscle mitochondria of normoxic and hyperoxic mice. Western blot was
hybridized with UCP3 antibody and subsequently with prohibitin antibody as described under Section 2. The expression of UCP3 is shown relative
to that of prohibitin. The ratio of the normoxic values is considered as 1.0. Results are expressed as means ± S.E.M. of arbitrary units; n = 11. (C)
Representative UCP3 and actin mRNA signals, and UCP3 and prohibitin protein signals are shown under the respective graph (Norm. = normoxia
and Hyp. = hyperoxia).\\, P < 0.005;\\\, P < 0.0005, unpaired Student?s t test.
Fig. 3. UCP3 mRNA expression and oxidative stress level in C2C12
myotubes under hyperoxia. (A) UCP3 mRNA levels in C2C12
myotubes under normoxic or hyperoxic conditions for 48 h. The levels
of UCP3 mRNA relative to those of cyclophilin were determined by
real-time PCR as described under Section 2. The ratio of the normoxic
values is considered as 1.0. (B) ROS level in C2C12 myotubes after
48 h of hyperoxia. Cells were loaded with 10 lM of CM-H2DCFDA
probe and ROS accumulation was measured as described under
Section 2. Values are means ± S.E.M. of six different determinations.
\\\, P < 0.0005, unpaired Student?s t test.
P. Flandin et al. / FEBS Letters 579 (2005) 3411–3415
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