Effect of Quercetin and DMSO on Skeletal Myogenesis from

C2C12 Skeletal Muscle Cells with Special Reference to PKB/Akt

Activity, Myogenin and Bcl-2 Expression
Arkadiusz Orzechowski(1), Katarzyna Grzelkowska, Wojciech Karlik and Tomasz
Motyl

(1) Department of Physiology, Biochemistry, Pharmacology and Toxicology, Fac-

ulty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland

Abstract
Conflicting data regarding the effect of antioxidants on skeletal myogenesis prompted us to
study the action of superoxide anion and hydroxyl radical scavengers on differentiating mu-
rine C2C12 myoblasts. The onset of myotube formation was delayed by quercetin and
DMSO while DNA synthesis was stimulated in response to elevated doses of both factors.
Cell viability measured by MTT assay was inhibited either by 100 µM quercetin or 1% or
2% of DMSO whereas elevated number of apoptotic cells was detected at the same time.
Muscle cell differentiation retarded by quercetin or DMSO was reflected by delay in myo-
genin expression and lowered distribution of myotubes with low (< 10) number of cell
myonuclei. For large myotubes (> 10) low scores for DMSO, and high scores for quercetin
were observed. Based on phosphorylation status, both antioxidants delayed PKB activation
and PKB-dependent differentiation, as well as antiapoptotic effect of PKB. Bcl-2 antia-
poptotic protein level was elevated earlier for control than for experimental treatment. Mu-
scle creatine kinase activity reflected the reduced rate of myogenesis. In conclusion, pro-
mitogenic activity of quercetin and DMSO disturbs differentiation programme of myoblasts
and might explain why more apoptotic cells was found after high doses of both factors. In
contrast to DMSO, quercetin-induced delay in myogenesis may result in larger muscle
mass. Results of this study support the idea that muscle differentiation can be regulated by
scavengers of superoxide anion and hydroxyl radical.
Key words: antioxidants, apoptosis, Bcl-2, muscle differentiation, myogenin, PKB.
Basic Appl Myol 11 (1): 31-44, 2001

Muscle growth results from hypertrophying myofi- impairs the survival of muscle precursor cells [53, 54].
bers, which increase their myonuclear number by re- With regard to transduction of signals carrying orders to
cruitment of satellite cells. Determined myoblasts, under survive, proliferate or differentiate attention is put on
conditions that favour differentiation, align and fuse to protein kinase B a product of Akt gene (PKB/Akt). PKB
form multinucleated myotubes [44]. The mechanisms protects against cell death either through downstream
underlying the recruitment of satellite cells to hyper- phosphorylation of Bad protein, subsequent release of
plastic or hypertrophic growth have not been estab- BclXL or Bcl-2 from heterodimers with Bad, sequestra-
lished. It is widely accepted, that high proliferation rate tion of Bax forming heterodimers with BclXL or Bcl-2,
delays the myogenesis and results in higher muscle and consequent inhibition of pore formation in the mito-
mass [8, 13, 37]. The higher the rate of cell multiplica- chondrial membrane which unable the release of cyto-
tion the higher the mass of growing muscle. Some chrome C (cyt C) and apoptosis inducing factor
growth factors and oncogenes as well as agents present (AIF)[36] or by phosphorylation-dependent inactivation
intracellularly or in the extracellular matrix (ECM) also of caspase 9 [15]. Two potent antagonists of reactive
can repress the activity of myogenin and subsequently oxygen species (ROS) action in muscle cells, namely,
inhibit myogenesis [5, 31, 51]. However, there is also quercetin and DMSO were evaluated in order to find the
evidence that accelerated mitogenesis in the circum- importance of these antioxidants to myogenesis. Quer-
stances the promote enhanced muscle differentiation cetin biological activities are pleiotropic, opposite to

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 Effect of quercetin and DMSO

ROS, and include cell specific inhibition of ubiquitous quercetin (larger myotubes). Both agents caused marked
enzymes (xanthine oxidase, tyrosine kinases, ornithine delay in PKB activation which was hardly followed by
decarboxylase (ODC), PI-3K, MAPK-s, calmodulin reduced rate of apoptosis; furthermore antiapoptotic
(CaM), lipooxygenase (LOX) and cyclooxygenase properties of quercetin and DMSO diminished with the
(COX) [10, 16, 17, 56]. The role of antioxidants in cell increasing dose of each factor. Every chosen biochemical
transformation is uncertain, although several groups of index of muscle cell differentiation (myogenin, MCK)
authors have reported that, for instance, once activated confirmed pictures obtained from microscopic observa-
by specific stimuli (inflammatory cytokines, ROS) he- tions (myotubes formed).
patic stellate cells differentiate into myofibroblast-like A deeper understanding of the role of phytochemicals
cells, while antioxidants including quercetin inactivate and thiols in the mechanism of muscle growth, devel-
this process [22, 23]. DMSO was well recognized for its opment and regeneration may have important implica-
ability to modulate the expression of various genes tions for the prevention and treatment of neuromuscular
negatively [7, 32] or positively inducing muscle cell dif- disorders. Further studies are needed to address the
ferentiation [2, 42]. However, up to date no data are above-mentioned effects and ascertain whether the
available to elucidate quercetin biological activity in physicochemical properties other then antioxidant cause
relation to muscle gene expression. According to studies delayed myogenesis.
on digestion and absorption, quercetin is the main die-
tary flavonoid consumed by Caucasians, so its relevance
Materials and Methods
for muscle development would be of great value [11]. In Reagents
rats fed diet enriched with 0.5% w/w blood plasma
All reagents were cell culture tested, of high purity,
quercetin may rise up to 100 µM [29], but in human
and unless otherwise stated they were purchased from
beings who consume approximately 50-80 mg of quer-
Sigma-Aldrich Chemical Co., plastics were from Corn-
cetin per day, the plasma level generally does not ex-
ing-Costar, while sera, media and antibiotics were from
ceed 1 µM [20].
Gibco Life Technologies. Phosphate buffered saline
Another equally important issue is the understanding
(PBS) and ultra pure agarose were obtained from Gibco
of abrogated myogenesis brought about by DMSO. Mu-
BRL. Primary rabbit polyclonal anti-Myogenin IgG an-
noz-Canoves et al. [34] showed that DMSO inhibited
tibody (M-225, sc-576), rabbit anti-Bcl-2 IgG antibody
myogenesis in cultured C2C12 myoblasts by repressing
(N-19; sc-492), and goat anti-Akt-1 IgG antibody (C-20;
urokinase type plasminogen activator (uPA) gene ex-
sc-1618) as well as fluorescein conjugated secondary
pression. Unfortunately, studies of Munoz-Canoves et
goat anti-rabbit IgG antibody for cytoimmunofluores-
al. [34] were not verified by the examination of cell sur-
cent studies were obtained from Santa Cruz Biotechnol-
vival. DMSO is a skin permeable popular vehicle and
ogy (Santa Cruz, CA, USA). Sodium dodecyl sulphate
compound used by body builders for topical treatment
(SDS) 10% (w/v), Sequi-Blot PVDF Membrane 0.2 µm
of the muscle pain and stiffness. It is also present in the
and all reagents for immunoblotting were obtained from
anti-inflammatory and anti-rheumatoid medicaments.
Bio-Rad Laboratories (CA, USA). Tris, EDTA, NaCl,
The issue whether and how DMSO influences muscle
proteinase K, RNAse A from bovine pancreas and
cell differentiation is a therefore a merit of considerable
Lambda DNA EcoR I HindIII Digest were obtained
importance.
from Sigma Chemical Co. (St. Louis, MO, USA). Other
Moreover, with regard to muscle cell differentiation it
reagents were purchased as stated in the description of
should be pointed out that delayed myogenesis with ex-
the respective methods (see below). Protein content was
tended period of cell multiplication but not apoptosis
assayed both by Lowry method (Sigma Chemical Co.
might be a characteristic feature of increased muscle
St. Louis, MO, USA) and the bicinchoninic acid method
mass. The aim of this paper was to provide a description
(BCA, Pierce Chemical Co., Rockford, IL, USA).
of the relation between the cell growth, formation of
myotubes, cell viability and apoptosis of cultured murine Cell culture
C2C12 muscle cells. Concomitantly, we scrutinized the Murine C2C12 myoblastic cell line (satellite cells
expression of myogenin, Bcl-2 protein, PKB and MCK from thigh muscle) purchased from European Collection
activity during differentiation. In this study we demon- of Animal Cell Cultures (ECACC) was maintained in
strated that antioxidants quercetin and DMSO delay the exponential phase of growth (20% (v/v) FCS/DMEM
expression of molecular and metabolic markers of differ- with Glutamax) designed as GM (growth medium) sup-
entiation. The results of our study suggest that superoxide plied with antibiotic-antimycotic mixture (Penicillin G
anion (quercetin) and hydroxyl radical (DMSO) scaven- sodium salt 50 IU/mL, Streptomycin sulphate 50
gers retard the onset of muscle cell fusion but, at least, in µg/mL; Gentamycin sulphate 20 µg/ml; Anti PPLO
the case of quercetin this effect was transient and once agent - Tylocine base 6 µg/mL, Fungizone - Amphoteri-
initiated myogenesis was significantly accelerated by cin B 1 µg/ml), in a controlled humidified air atmos-

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 Effect of quercetin and DMSO

phere supplied with 5% CO2, at 37°C in a multiwell or 33342) and propidium iodide (PI) as described by Abu-
tissue culture Petri dishes (Corning-Costar Inc. U.S.A.). Shakra et al. [1]. Percentage of apoptotis-apoptotic index
(the number of apoptotic nuclei expressed per total num-
Experimental procedure
ber of nuclei) was calculated. Multiwell (6) dishes were
Every other day the cells were washed twice with phos- used to grow myoblasts on cover slips coated with a 10%
phate buffered saline (PBS) and medium was changed gelatine film. Experimental media were added, and at the
until they reached 100% confluence. Confluent cells end of each following 24 hours of study fluorochromes
(myoblasts of the same cell density fully covering surface (HO 33342 or PI) were added. Firstly, for the last 30 min.
dish) were then guided to post mitotic status, and differ- bisbenzimide HO 33342 (stock solution of 25 mg/mL in
entiation and fusion were initiated by replacing GM with H2O) was introduced to give final concentration of 0.3
2% (v/v) horse serum HS/DMEM designed as DM (dif- mg/mL, then for the final 5 min. propidium iodide (stock
ferentiating medium). In the above mentioned conditions solution of 10 mg/mL in PBS) was added to give working
C2C12 myoblasts easily and fully differentiate into solution of 5 µg/mL. Media were aspirated, cells gently
myotubes, therefore we could follow up modifications of washed with ice cold PBS, and mounted on slides using
differentiation process during 5 subsequent days. During mounting medium anti-fading solution (DAKO, Den-
the study freshly prepared media without or with the ex- mark). Since PI does not enter cells that are alive, dead
perimental factors were changed every 24 hours. Querce- cells (late apoptotic and necrotic) were stained with this
tin dissolved in DMSO (1, 10, 100 µM) or DMSO (0.1, 1, fluorochrome. On the other hand, HO 33342 penetrates
2% v/v) were added to the medium. With regard to quer- every cell and also stains nuclear DNA. Fluorescent mi-
cetin, the lowest DMSO concentration present (0.1% v/v) croscope BX-60 Olympus equipped with a PM20 auto-
played the role of the control system, while for DMSO matic photomicrograph system was used for photo-
treatments fresh DM medium became the control system graphic recording. In the ultraviolet light at least one
additionally shown on figures. thousand nuclei in total were counted in ten (or more if
Cell viability necessary) randomly chosen visual fields per each slide
Assessment of cell viability based on mitochondrial and cells were qualified as follows: regular oval shaped
function was assayed by the ability of cells to convert blue nuclei (alive cells); condensed white-blue nuclei
soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl- (apoptotic). In the same visual field the excitation of
tetrazolium bromide) into an insoluble purple formazan propidium iodide light led to the appearance of red nuclei
reaction product with minor modifications to protocol of dead cells, mainly apoptotic.
described by Jacobson et al. [21]. The GM medium was Proliferation and protein assays
replaced with DM medium with or without experimen- Simultaneous labelling with (3H)-methyl-thymidine
tal factors (changed every 24 h), and for this assay dur- and (14C)-leucine (Amersham PLC, 1 µCi/mL, 5
ing the last 4 h of incubation time of each day these me- Ci/mmol) was used every day during 5 days (24 h of
dia were replaced with MTT solution (0.5 mg/mL in treatment) in order to determine changes in both the
DMEM without phenol red, sterilised by filtration). proliferation assay (mitogenicity) and protein synthesis
MTT solution was then aspirated and formazan was dis- (anabolism). When cells became fully confluent, cul-
solved by addition of 100 µL dimethylsulfoxide tures were washed twice with PBS and this day was set
(DMSO). The absorbance (570-630 nm) was measured as day “0”. Control and experimental media with quer-
with ELISA Reader type ELx808, BIO-TEK Instru- cetin and DMSO were poured into multiwell (24)
ments (U.S.A) and % survival was defined as ((experi- plates. A volume of 10 µL of mixture of 30 µCi/mL of
mental-blank)/(control-blank)) x 100, where the blank each label, to give final concentration of 1 µCi/mL of
was the value obtained from wells containing DMSO the radioisotope mixture was added to the one of the
without cells. In all cases, the cells were examined un- plates and the plates were transferred to the incubator
der phase-contrast microscopy before application of as described for cell culture. Total label time was 24
MTT to visually assess the degree of cell death. Percent hours. Every day cells were washed twice with PBS
viability (MTT conversion into purple formazan in and immersed with fresh media, whereas one plate was
comparison with control value of 2% HS/DMEM for supplemented with a radioactive label. After 24 h in-
DMSO or 0.1% DMSO in 2% HS/DMEM for querce- cubation with the label the cells were subject to fixa-
tin) indicates cell viability (mitochondrial respiration or tion. The wells were poured with 0.5 mL of TCA (10%
activity of mitochondrial dehydrogenases). w/v) to precipitate protein and DNA. After overnight
Apoptosis incubation at 4°C, TCA solution was aspirated and the
Cytotoxicity with resultant cell death was monitored by cells were washed for 5 min with 70% v/v methanol
microscopic observations (Olympus BX-20). Apoptosis followed by a 5 min wash with 90% v/v methanol. The
was evaluated by in situ uptake of bisbenzimide (HO cells were dissolved in 0.25 mL of 0.5 M NaOH sup-
plied with 0.2% v/v Triton X-100, kept in the incuba-

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 Effect of quercetin and DMSO

tor for 24-48 h (37°C, 95% humidity). The completion goat antibody conjugated with horseradish peroxidase.
whether the cells were dissolved was controlled micro- The blots were developed using the enhanced chemi-
scopically. Soluble cell solution was neutralised with 5 luminescence (ECL) detection system (Amersham) ac-
M formic acid, mixed with scintillation cocktail cording to the manufacturer’s protocol. The mem-
(Aquasol, New England Nuclear), vigorously agitated, branes were scanned and analysed using a JX-330
and counted in Packard TRI-CARB 1600 CA β- Sharp scanner and densitometry of bands was per-
counter. The results obtained in d.p.m. were expressed formed using Diversity OneTM version 1.3 software
in arbitrary units as % values (experimental values at (pdi, New York, NY, USA).
the following days divided by the control value at day
Cytoimmunofluorescence studies
1. of experiment multiplied by 100).
The presence and intracellular location of certain
Myotube formation regulatory proteins was based on the immunocyto-
Cell cultures were also analysed morphologically after chemical detection performed by two-step reaction. In
fixing and staining with Giemsa reagent. Mononuclear 6 well (35 mm diameter) tissue culture dishes cells
myocytes (spindle shaped) and multinuclear (3-10 myo- grown on cover slips coated with 10% gelatine were
nuclei per myotube) as well as (10 > myonuclei per fixed with methanol free 1% (w/v) formaldehyde in
myotube) myotubes were identified with a contrast- PBS for 15 min (37°C, 100% humidity), washed twice
phase microscope (Olympus BX-20) in 10 randomly with PBS and transferred into 70% ethanol (stored up
chosen microscopic fields and photographed. Since the to 48 h at -20°C). Subsequently, the cells were washed
number of cells does not increase in confluent cultures twice with PBS supplemented with 1% (w/v) bovine
myotube indices (3 > 10 myonuclei per myotube) and serum albumin (PBS-BSA). The cells were than im-
(10 myonuclei > per myotube) were calculated by scor- mersed under 100 µl of sterile primary rabbit anti-
ing the number of myotubes containing certain number Myogenin or anti-Bcl-2 IgG antibody solution (10
of myonuclei present per microscopic field. µg/ml w/v, Santa Cruz, CA, USA) in 1% PBS-BSA
and incubated for 1.5 h (37°C, 100% humidity). Rabbit
Creatine kinase activity
anti-IgG antibody (Santa Cruz, CA, USA) served as
Muscle creatine kinase (MCK) activity in cell lysates isotype negative control. The cells were washed twice
was determined using CK assay kit (Sigma Diagnostics, with PBS, immersed under 100 µl of secondary FITC-
St. Lois, MO, USA) with N-acetyl cysteine included as conjugated goat-anti rabbit IgG diluted 1:100 v/v
the reagent to stimulate MCK activity. (Santa Cruz, CA, USA) and incubated again for 1.5 h
Immunoblotting (37°C, 100% humidity). After a subsequent double
Immunoblotting was performed as follows: an ali- wash with PBS cover slips were drained and mounted
quot of ice-cold extraction buffer containing: 50 mM upside down on microscopic slides covered with a
Tris-acetate, 50 mM NaF, 2.5 mM EDTA, 1 mM drop of mounting medium (DAKO Corp. Denmark).
EGTA, 5 mM sodium pyrophosphate, 5 mM β- Sometimes the myonuclei were labelled with Actino-
glycerophosphate, 1 mM sodium orthovanadate mycin D (5 µg/ml H2O2, 10 min, 4°C) before micro-
(Na3VO4), 2 mM dithioerytreitol (DTT), 1 mM ben- scopic evaluation. The cells were observed under a
zamidine, 4 µg leupeptin, and 1% (v/v) SDS, pH 7.2) fluorescent microscope and photographed (fluorescent
was added to a tube and the cellular suspension was microscope BX-60 Olympus equipped with the PM20
dissolved by repetitive triturating with a pipette tip and automatic photomicrograph system).
left to stay for 10 min at room temperature. Viscous Oligonucleosomal fragmentation of DNA
solution was then transferred to a fresh tube, frozen in The method for agarose-gel electrophoresis of whole
liquid nitrogen and stored at -70°C until used. Western cells described by Wolfe et al. [55] was adopted with
blot analysis was carried out using equal amounts of minor modifications. Briefly, approximately 1 x 106 of
protein (100 µg) subjected to SDS-PAGE under re- cells grown on tissue culture Petri dishes (35 mm di-
ducing conditions. Electrotransfer of proteins to PVDF ameter) was aseptically harvested with rubber police-
membranes (0.2 µm) was performed for 1 h at 100 V man in PBS-D, monolayer and medium transferred
and followed by blocking in TBS buffer (20 mM Tris, into sterile Eppendorf tubes and centrifuged (800 g, 10
500 mM NaCl, pH 7.5) supplemented with 5% non-fat min, 4°C), the supernatant was aspired, and the cell
powdered milk. The membranes were then probed with pellet after wash with PBS and concomitant centrifu-
primary antibody - rabbit polyclonal anti-Myogenin (1 gation was held in sterile Eppendorf tubes until assay
µg/ml), rabbit polyclonal anti-Bcl-2 (1 µg/ml, ) and (4°C). The cells were resuspended by triturating in 15
goat polyclonal anti-Akt-1 (1 µg/ml) for 16 h at 4°C, µL of sterile deionized water, vortexed for 1 sec. Ap-
washed three times in TBS containing 0.05% Tween- proximately 6 µL of RNAse A solution (10 mg/mL in
20 and were incubated with goat anti-rabbit or anti- 10 mM Tris, 15 mM sodium acetate, pH 7.5, pre-

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 Effect of quercetin and DMSO

heated for 15 min in 95°C) was added to each tube, the points the results were analysed by two-way analysis
samples were mixed by spin and incubated at 56°C for of variance. The results were expressed as the means
1 h. Afterwards, 9 µL of loading buffer (12% Ficoll and SEM with a value of P < 0.05 taken as significant,
400 in TAE, 0.25% bromophenol blue and 0.25% xy- P < 0.01 as highly significant and P < 0.001 as very
lene cyan blue) was added and the samples were spun highly significant.
again. While running digestion, the gels were prepared
by dissolving 1.8 g agarose in TAE buffer 1.8% (w/v)
Results
(10 mM Tris, 10 mM sodium acetate, 1 mM EDTA) Proliferation assay and protein synthesis
and melted it in a microwave oven. 1 ml of 10% SDS
Simultaneous determination of both DNA synthesis
(w/v) was added to liquid gel to give the final content
and protein synthesis provide indirect information
of 2% followed by addition of 20 µL of proteinase K
about the rate of cell growth occurring during tissue
(20 mg/ml) after cooling to 55°C. The solution was
development [38]. In the control system (DM) the
then immediately poured into the gap above the wells.
slope of curve representing time-course for multiplic-
Each sample was transferred into each well. 1 µL of λ
ity (proliferation assay - DNA synthesis) was falling
DNA EcoR I HindIII Digest (125-21,226 bp) mixed
(by 74% in extreme; P < 0.001, Fig. 1a), whereas the
with 10 µL TAE and 5 µL of loading buffer was ap-
slope of curve illustrating time-course of protein syn-
plied to each gel to provide a size marker. Electropho-
thesis was elevating (by 70% in extreme; P < 0.001;
resis of DNA was initially performed with 2V/cm (20
Fig. 1b).
Volts) for 1 h, followed by 3 h with 8V/cm (80 Volts)
Quercetin and DMSO, regardless of the dose used,
with TAE as running buffer. After electrophoresis the
were apparently mitogenic (P < 0.001). However,
gels were stained for 20 min with ethidium bromide (1
DNA synthesis rate was not in direct proportion to the
µg/mL) followed by 5 min washing with deionized
concentration of DMSO or quercetin (Fig. 1a). Mito-
water. The gels were then UV illuminated and photo-
graphed by Biometra BioDoc II video imaging system genicity stimulated by quercetin, in extremes,
(U.S.A.). amounted to 50%, 58%, and 12% for 1, 10 and 100
µM, respectively, whereas for DMSO reached 20%, 0,
Statistical analysis 40% for 0.1%, 1% and 2%, respectively. Also protein
Value on day 1. in the control group for each series synthesis did not directly respond to the dose of ex-
of treatments was set as 100%. All remaining data perimental factors (P < 0.001). For quercetin, protein
(unless otherwise stated) were expressed as % of this formation was inhibited by 12% after 1 µM (P < 0.05)
value (% of control). Since quercetin was dissolved in and by 60% after 100 µM (P < 0.001), except stimula-
DMSO (present as 0.1% v/v of experimental medium), tion by 80% after 10 µM of quercetin (Fig 1b;
0.1% of DMSO is to be chosen as a control group for P < 0.01). This relation was especially evident for
quercetin. The statistical analysis was performed by DMSO, where protein synthesis rate did not differ
one-way analysis of variance. Determination of sig- from control after 0.1% DMSO, whereas it was inhib-
nificance between the differences of the means was ited after 1% and 2% of DMSO, respectively (Fig. 1b;
carried out with Tukey’s multiple range test. In order P < 0.001).
to compare the treatment means at the same time

Figure 1. Effects of various concentrations of quercetin or DMSO on DNA synthesis (proliferation assay) (a) or pro-
tein synthesis (b). Confluent murine C2C12 myoblasts grown on multiwell (24) plates were incubated in DMEM
supplemented with 2% HS (DM) in the absence or presence of quercetin (1, 10, 100 µM) or DMSO (0.1, 1, 2%
v/v) for 5 days. At each time point (day 1., 2., 3., 4., 5.), cells from three wells of multiwell plate per each treat-
ment taken and radioactivity (d.p.m.) of TCA precipitable fractions of (3H)-thymidine labeled DNA and (12C)-
leucine labeled protein were assessed as indicated. The experiment was repeated at least twice with similar re-
sults. Values are means with SEM.

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 Effect of quercetin and DMSO

Figure 2. Effects of various concentrations of quercetin or DMSO on myotube indices (3-10 myonuclei per myotube)
(a) or (10 > myonuclei per myotube) (b). Confluent murine C2C12 myoblasts were incubated in DMEM sup-
plemented with 2% HS (DM) in the absence or presence quercetin (1, 10, 100 µM) or DMSO (0.1, 1, 2% v/v)
for 5 days. At each time point (day 1., 2., 3., 4., 5.), cells from three wells of multiwell plate per each treatment
were fixed and stained with Giemsa and myotubes counted as indicated. The experiment was repeated at least
twice with similar results. Values are means with SEM.
sequent days, the expression of myogenin was signifi-
Myotube formation
cantly accentuated in growing myotubes both in con-
Since the intensity of myotube formation may differ, trols and after quercetin and DMSO (P < 0.001). In
we decided to study the effect of each of the experi- great part the results of immunobloting were consistent
mental agent on the presence of either myotubes with with cytoimmunofluorescent studies. The blots repre-
3-10 myonuclei (initial step of fusion) or myotubes senting the myogenin protein concentration showed a
with 10 or more myonuclei (late step of fusion), sepa- time-dependent increase in density (Fig. 3). The blots
rately. illustrating myogenin proved the inhibitory effect of
Both quercetin and DMSO were observed to delay the both antioxidants on the expression of myogenin gene.
onset of myotube formation ( < 10 myonuclei) in com-
parison to controls (Fig. 2a, P < 0.001). Also the number
Muscle creatine kinase activity
of myotubes formed after the replacement of GM with The activity of MCK rose with the time of incubation
DM was higher in controls than in the experimental (P < 0.001), but in relation to the time of collection (1.,
cultures (P < 0.001). While quercetin was shown to ac- 3., 5. day) the highest values of the mean were observed
celerate the formation of already existing myotubes for controls, lower for quercetin and the lowest for
(10 > myonuclei) manifested by more numerous large DMSO (Fig. 4, P < 0.01). Elevated activity of MCK
myotubes (Fig. 2b; increase by 250%; P < 0.001), corresponded to the accelerated respiration of myotubes
DMSO was found to maintain inhibitory effect until the determined by MTT test.
very end of experiment (Fig. 2b; decrease by 250%; PKB activity
P < 0.001). Furthermore, during 2% DMSO treatment Once activated, protein kinase B has phosphorylated
no myotube with more than 10 myonuclei was found all form, therefore the additional upper band of PKB of
over the experimental period. Both effects were time- higher molecular weight is found. The density of dot
dependent (Fig. 2). blots is directly proportional to the extent of phosphory-
Myogenin expression lation. During 5 days of induced myogenesis, separated
and established identity of PKB was found to be both free
Activation of myogenin gene was monitored in the
form and phosphorylated form, with the remark that after
aim to find out whether morphological alterations in
DM phosphorylation of PKB reached maximum on day
the size and nuclei content of myotubes are mirrored
2. (Fig. 5). Then the activation gradually lessen. Querce-
by the transcriptional activity of the important mo- tin and DMSO delayed PKB phosphorylation, so the
lecular inductor of E-box dependent muscle specific blots of phospho-PKB were observed during subsequent
genes. Myogenin expression was examined either by days with the maximum on day 4 (Fig. 5).
cytoimmunofluorescence or by immunobloting. After
quercetin or DMSO, we observed microscopically a Apoptosis
marked gap (24 h) of myogenin expression, which re- Differentiation and fusion of myoblasts might be at-
flects the absence of myotubes at the same time (data tenuated by other mechanisms than influence on signal
not shown). For the first time myogenin protein had transduction and gene expression. A good example is
been observed by immunoblotting at the onset of illustrated by the fact that cells, which undergo the cell
myotube formation - after 24 h for controls, and after cycle exit or those, which are on the way to fusion
48 hours for the experimental treatments. During sub- process in certain circumstances can be deleted (pre-

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 Effect of quercetin and DMSO

appear by the mechanism of cell death during differ-

entiation well documented as cited below. Consistently
with the previous reports [1, 15, 45, 48, 53, 54] apop-
tosis of mononuclear myogenic cells was exaggerated
with the time of differentiation reaching the maximum
on day 4. (increase from 1% on day 1. to 8% on day 5.,
or from 2% on day 1. to 9% on day 5.; P < 0.001, Fig.
6a). In comparison, both antioxidants at low level were
less apoptotic, so AI at last was equal to controls, but
elevated i.e. for 2% DMSO. Therefore, the less apop-
togenic activity of quercetin and DMSO was adversely
proportional to the dose of each agent. Quercetin at 1
µM inhibited apoptotic cell death (0-2% at maximum;
P < 0.001), whereas a further increase in concentration
caused elevation to 7% at maximum (P > 0.05) (Fig.
6a). After 1 day of the experiment very similar repre-
sentation of condensed nuclei was observed for DMSO
treatments, approximately 3% vs. 1% of control
(P < 0.05). At the maximum, AI amounted to 8% for
control and almost to 9% for 2% of DMSO (P < 0.05).
The concentration of 1% and to a higher degree 0.1%
of DMSO led to a very highly significant reduction in
AI value. Since the AI reached maximum on day 4. of
experiment (P < 0.001), we decided to check whether
fragmented DNA was present in cell cultures treated
Figure 3. The results of densitometric analysis (optical with either of the antioxidants. Both quercetin and
density, OD x mm2) of myogenin (filled bars) ex- DMSO caused the presence of small molecular weight
pression during differentiation of C2C12 myo- DNA fragments but the formation of oligonucleosomes
blasts. Confluent murine C2C12 myoblasts was not observed, although smears of lysed DNA were
grown on Petri dishes (60 mm diameter) were found for 1% and 2% of DMSO (Fig. 7). It seems, that
incubated in DMEM supplemented with 2% HS both antioxidants at high doses directly or indirectly
(DM) in the absence (Control); or the presence inhibited viability of the cells.
quercetin (100 µM Quercetin) or DMSO (1% Viability of the cells
DMSO). At each time point (day 1., 2., 3., 4., 5.),
plate of cells from each day was taken and pro- In order to examine whether quercetin and DMSO
teins were analyzed by SDS-PAGE followed by disturb vital processes in the cells, we carried out a se-
Western blot. The results are representative of ries of viability tests (MTT assay). The reduction of
the experiments that were repeated three times MTT into insoluble formazan occurs exclusively at the
with similar results. expense of reducing equivalents, the essential condi-
tion of life supported by energy demands. In control
mature mitosis or mitotic catastrophe). During apopto- systems the viability rose with time up to 40% above
sis cell membrane remains intact, chromatin is con- the reference value (P < 0.001). Quercetin at 1 and 10
densed and finally cell blabbing occurs with the cell µM did affect cell viability by stimulating cellular res-
fragmentation to apoptotic bodies. Regardless of the piration (15% above control, P < 0.01), but at 100 µM
route of cell elimination, the essential question is the process of a progressive increase in MTT reduction
whether dying cells are those, which are currently was inhibited (by 10% on day 2., P < 0.05) and re-
forming myotubes, or others, which are not involved in mained unchanged until the end of the study (Fig. 6b).
the fusion process. Observations performed on cells On the other hand, for DMSO we found dose- and
simultaneously stained in situ with HO33342 and PI time-dependent loss of viability, slight for 0.1% (re-
enable identification of floating cells, mononuclear duction by 14%; P < 0.05), but higher for 1% (85%;
cells and myonuclei marked with dense chromatin, a P < 0.001), and 2% (100%; P < 0.001) at the end of the
symptom of apoptotic cell death. The apoptotic index examination period. Actually, restricted respiration
(AI) was calculated, and a photographic record per- was concomitant with the elevated rate of apoptosis,
formed to visualize the necrobiology of the muscle and for quercetin the cells that were alive respired with
cells. Assuming the lack of injury, the cells might dis- higher intensity than controls.

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 Effect of quercetin and DMSO

Figure 4. Effects of various concentrations of quercetin

or DMSO on muscle cell creatine kinase (MCK)
activity. Confluent murine C2C12 myoblasts
grown on in Petri dishes (60 mm diameter) were
incubated in DMEM supplemented with 2% HS
(DM) in the absence or presence of quercetin (1,
10, 100 µM) or DMSO (0.1, 1, 2% v/v) for 5
days. At each time point (day 1., 3., 5.), three
plates of cells from each treatment combination
were taken and MCK activity was determined as
indicated. The experiment was repeated twice
with similar results. Values are means with SEM.

Bcl-2 expression Figure 5. The results of densitometric analysis (optical

Bcl-2 protooncogene product plays the key role in density, OD x mm2) of PKB (filled bars) and
preventing apoptosis by sequestrating other apopto- PKB-P (open bars) expression during differen-
tiation of C2C12 myoblasts. Confluent murine
genic proteins of the same family i.e. Bax, Bid [26].
C2C12 myoblasts grown on Petri dishes (60 mm
Since the interplay between these proteins directly de-
diameter) were incubated in DMEM supple-
termines the occurrence of cyt C-dependent apoptosis
mented with 2% HS (DM) in the absence (Con-
either expression or intracellular translocation of Bcl-2
trol); or the presence quercetin (100 µM querce-
family proteins is regarded as an important determi-
tin) or DMSO (1% DMSO). At each time point
nant illustrating susceptibility of cells to apoptogenic (day 1., 2., 3., 4., 5.), plate of cells from each day
signals. By immunobloting, reasonable similarities was taken and proteins were analyzed by SDS-
between the expression of Bcl-2 protein and PKB acti- PAGE followed by Western blot. The results are
vation were observed. During myogenesis Bcl-2 pro- representative of the experiments that were re-
tein peaked on day 2. whereas after quercetin or after peated three times with similar results.
DMSO treatment Bcl-2 protein level was elevated on
day 4. (P > 0.05; Fig. 7). A similar picture was demon- thyronine (T3) of activating protein (AP-1) transcrip-
strated on the basis of cytoimmunofluorescent studies. tion complex via the suppression of redox sensitive 9-
cis-retinoic acid receptor (RXR) mediated by cAMP
Discussion pathway [30] and B-cell translocation gene 1 antipro-
Up to date some factors that posses antioxidant ac- liferative protein (BTG1) [41] led to enhanced muscle
tivity or modulate intracellular redox status have been cell differentiation. In contrast studies performed with
extensively studied and compared with regard to myo- vitamin C derivative discovered its promoting action
genic differentiation. Down-regulation of myogenin on muscle cell differentiation. After ascorbic acid 2
expression and myotube formation was described for phosphate (Asc-2-P) the degree of fusion content in
aspirin in quail myoblasts [18]. Aspirin and/or salicy- the culture was similar to control except the larger di-
lates are antioxidants, which inhibit oxidation initiated ameter of myotubes. However, the mechanism of in-
by superoxide/nitric oxide radicals [18], as well as in- creased differentiation after Asc-2-P was indirect,
hibit experimentally induced lipid peroxidation and since no expression of molecular and biochemical
DNA damage [47]. When the hormone target is lo- markers of myogenesis were observed after imple-
cated at nuclear level (steroids or thyroid hormones) mentation of specific collagen synthesis inhibitor [33].
the direct transcriptional interplay between the nuclear On the other hand, retinoic acid and its derivatives ei-
receptors might also influence myogenesis [9]. For ex- ther block both growth and fusion of L6 myoblasts
ample, well characterised down-regulation by triiodo- [49], or stimulate myogenesis in C2C12 myoblasts by
down-regulating the expression of syndecan-1, ECM

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 Effect of quercetin and DMSO

Figure 6. Effects of various concentrations of quercetin or DMSO on apoptotic index (AI) (a) or cell viability (MTT
test) (b). Confluent murine C2C12 myoblasts were incubated in DMEM supplemented with 2% HS (DM) in the
absence or presence quercetin (1, 10, 100 µM) or DMSO (0.1, 1, 2 % v/v) for 5 days. At each time point (day 1.,
2., 3., 4., 5.), cells from three wells of multiwell plate per each treatment were fixed and all nuclei stained with
HO 33342, as well as nuclei with condensed chromatin stained both with HO 33342 and PI were counted per
microscopic field to assess the fraction of apoptotic nuclei or treated with MTT reagent. The experiment was
repeated at least twice with similar results. Values are means with SEM.
proteoglycan involved in the antimyogenicity exerted guess that after reduced serum (DM) both antioxidants
by such mitogens as bFGF and TGF-β1 [28]. might act as survival factors when used at low concen-
Furthermore, for instance DMSO has been long trations. At the same time cell death machinery was
known [7, 32] to almost completely abrogate myogene- markedly hampered, but with increased doses the out-
sis. Recently it has been demonstrated that DMSO acts comes of aberrant myogenesis led to the activation of
through the mechanism of transcriptional repression of apopoptotic cell death. The lack of DNA ladder could
uPA, that is the moderator of structural reorganisation therefore be a characteristic sign of apoptosis induced
of ECM that precedes the fusion of myoblasts [34]. by antioxidants or the result of cells washed off the
Myogenin expression is also repressed by sodium bu- DNA fragments in differentiating C2C12 myoblasts.
tyrate, routinely used to block the checkpoint at the end PKB/Akt is the main target of insulin signalling and
of MyoD transcription family cascade [27]. Taken to- regulation of cell survival [52]. Among the targets of
gether, careful analyses of the results obtained from the PKB that delay cell death are Bad, caspase 9 and IKKs
studies with factors that express anti- or prooxidant be- [15, 36]. As it was mentioned in the introduction, upon
haviour have shown that myogenesis is often moderated phosphorylation of Bad, antiapoptotic proteins Bcl-2 or
by redox dependent mechanisms. BclXL are no longer bound to Bad and can effectively
PKB activation accompanies discontinuing of cell divi- dimerize with Bax preventing the execution of apoptosis.
sions, hence protects differentiating myoblasts against Likewise, when PKB phosphorylate caspase-9, the en-
death caused by the cessation of survival signals normally zyme is inactivated and the late phase of apoptosis is ab-
approaching the cells during proliferation but not if myo- rogated. New findings show that Akt is also involved in
genin is to be activated. The perturbation in cell cycle ac- lowering the expression of Fas ligand, which by acting on
tivity or during cell cycle exit at the differentiation phase Fas/Apo1 receptor (family of TNF-α receptors) induces
may lead to extensive death of myoblasts (see review by cell death. The mechanism of such antiapoptotic action of
Sandri and Carraro [43]). Actually, from studies per- PKB resulted from the phosphorylation of fork head re-
formed on cell lines and animals either deficient or with lated transcription factors (FKHR, FKHRL1), which
forced expression of certain genes, growing evidence translocate into cytosol, became sequestrated by 14-3-3
proves that in fact muscle cell deletion is exacerbated af- proteins and consequently are unable to target nuclear
ter interrupted expression of protooncogenes [50, 57]. genes coding for Fas ligand [14]. Although in general a
During our studies an increase in apoptosis by querce- variety of apoptogenic signals are potentially blocked by
tin (100 µM) or DMSO (1,2%) was observed, although PKB, overexpression of Akt does not fully protect mitotic
at low doses both antioxidants apparently inhibited C2C12 myoblasts from apoptosis during myogenesis
apoptosis. Anyway, we could not demonstrate oligonu- [15]. This was also the case in our study, when PKB was
cleosomal fragmentation of DNA after treatment with at last activated either in the presence of quercetin or
quercetin and DMSO despite the presence of apoptotic DMSO, the antiapoptotic activity diminished with ele-
phenotype. Taken together, these findings and other vated doses of each agent. It is most likely that other al-
from different laboratories indicate that, at least, a few ternative antiapoptotic mechanism might explain the
alternative apoptotic pathways may exist [6, 24]. We presence of apoptosis after activation of PKB.

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 Effect of quercetin and DMSO

Figure 8. The results of densitometric analysis (optical

density, OD x mm2) of Bcl-2 (filled bars) expres-
sion during differentiation of C2C12 myoblasts.
Confluent murine C2C12 myoblasts grown on
Petri dishes (60 mm diameter) were incubated in
DMEM supplemented with 2% HS (DM) in the
absence (Control); or the presence quercetin
(100 µM Quercetin) or DMSO (1% DMSO). At
Figure 7. Effects of various concentrations of quercetin each time point (day 1., 2., 3., 4., 5.), plate of
or DMSO on the presence of DNA fragmentation cells from each day was taken and proteins were
in cell lysates. Confluent murine C2C12 myo- analyzed by SDS-PAGE followed by Western
blasts grown on in Petri dishes (60 mm diameter) blot. The results are representative of the ex-
were incubated in DMEM supplemented with 2% periments that were repeated three times with
HS (DM) in the absence or presence quercetin similar results.
(1, 10, 100 µM) or DMSO (0.1, 1, 2% v/v). On
day 4. formation of oligonucleosomal DNA in The results obtained from our studies are consistent
each well was assessed as indicated. The ex- with the statement of time-dependent elevation in phos-
periment was repeated at least twice with similar phorylation status of threonine/serine PKB during myo-
results. Approximately 1x106 cells from each genesis. Actually, differentiation of C2C12 myoblasts is
treatment were taken for analyses. The cells were associated with the activation of PI-3K, an indirect up-
washed twice with PBS-D and subjected for fur- stream activator of Akt [42, 43], similarly to other myo-
ther preparation as described. Lanes signed with genic cell types [19, 25]. However, stimulation of
subsequent numbers (1-7) represent: (C) control, PKB/Akt activity by 100 µM of quercetin and 1%
(1, 2, 3) increasing quercetin doses (1, 10, 100 DMSO, observed in our experiment, was delayed and ac-
µM), (4, 5, 6) increasing doses of DMSO (0.1, 1, companied by symptoms of muscle differentiation. This
2% v/v, respectively). Capital letter M stands for may suggest that stimulatory signals probably originated
λ DNA EcoR I HindIII Digest (125-21,226 bp). from antioxidants and directed on PI-3K phosphorylation
were retarded; therefore subsequent myogenesis was de-
pendent on PKB downstream-regulated cascade. There
are some striking parallels between the results presented

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 Effect of quercetin and DMSO

here and those previously reported by others [7, 32]. translocation among the members of Bcl-2 family or
Our data indicate that cell elimination was a significant other apoptogenic/antiapoptotic proteins does not
factor, which contributed markedly to determine abro- merely accounted for the C2C12 apoptosis. PKB activa-
gated myogenesis by DMSO. These observations are tion was positively correlated with PKB phosphorylation
likely to have direct bearing on the role of factors af- and Bcl-2 expression.
fecting muscle cell death and restricted differentiation. Anyway, the requirement for satellite cell proliferation
In this scenario, DMSO would negatively regulate myo- and differentiation to support muscle hypertrophy indi-
genesis and accelerates apoptosis of muscle cells. Quer- cates that a transient delay in myogenesis after quercetin
cetin activity on myogenesis differed from DMSO. A and possibly other mitogenic antioxidants might also
delay in muscle differentiation by quercetin was found play a putative role in the positive regulation of muscle
at high dose whereas sustained DMSO-induced arrest of growth. Just recently, some important observations were
myogenesis was observed. Later on, the already formed drawn by Puri et al. [39] who observed induction of dif-
myotubes grew faster after quercetin than in controls. ferentiation of human rhabdomyosarcoma cells into
Our results suggest that antioxidant properties of quer- fully developed myotubes by p38 MAP kinase which is
cetin and DMSO should be responsible for similarities well known to be activated by ROS. Similarly, activa-
observed between affected muscle cells merely during tion of mitochondria due to the higher activity of com-
initial 24 h of experiment. plex I releases of H2O2 (Barja 2000) also led to differ-
We have demonstrated that apoptosis of C2C12 mus- entiation of chick myoblasts [40]. The mechanisms un-
cle satellite cells increased during myogenesis with the derlying the recruitment of satellite cells for regenera-
maximum on day 4. We also provided evidence that tive or hypertrophic processes have not been estab-
quercetin and DMSO were less apoptotic, but this prop- lished, although the role of various growth factors until
erty disappeared with elevated doses of both agents. In now was thought to be crucial [4, 35]. However, most
this regard, cellular respiration diminished with in- mitogenic growth factors have been found to inhibit dif-
creasing rate of apoptotic cell death. However, when ferentiation and fusion, and what we have found for the
curves representing time-course of viability and apopto- first time, also by antioxidants such as quercetin and
sis in control systems were plotted against each other, DMSO. The course of changes in markers of myogene-
striking similarities appeared. No dissociation between sis is sequential with the myogenin gene product being
the slopes of curves representing cell viability and the ultimate determinant of molecular commitment of
apoptosis suggests that myotubes adapted to a higher myoblasts to myogenesis. It would be advantageous to
energy demand by increasing activation of mitochon- verify the working hypothesis if quercetin and DMSO
drial dehydrogenases to the extent that could overcome directly or indirectly affect myogenin gene expression,
the loss of reducing equivalents from deleted cells. Such and whether other kinases with possibly equal impor-
assumption is strongly supported by elevated MCK ac- tance as PKB in Bad phosphorylation are PKA (which
tivity observed during myogenesis. However, this was also inhibits myogenesis) and MAP kinases. Further
not the case for DMSO indicating that aberrant myo- work is required to understand the physiological rele-
genesis was linked to the failure of respiration of C2C12 vance of the modified redox status within the muscle
myoblasts to execute a differentiation programme. With cells.
regard to quercetin merely modest inhibition of muscle In summary, our findings suggest that superoxide an-
differentiation was shown (day 1.). Taken together, ion (quercetin) and hydroxyl radical (DMSO) scaven-
these observations indicate the possibility that antioxi- gers influence myogenesis. We observed suppressed
dants with mitogenic activity at high doses might in myogenin expression, delayed activation of PKB/Akt
some way trigger the apoptotic cell death of differenti- and Bcl-2 expression, decreasing antiapoptotic effect of
ating cells. Moreover, these cells cannot be rescued by both agents with concomitant stimulation of mitogenic-
concomitant activation of PKB/Akt. Additionally, it is ity and reduced anabolism. Less numerous but larger
conceivable that DMSO at most abrogated myogenesis myotubes found at the end of the experimental period
partially due to the elimination of differentiating myo- after quercetin could be considered as a sign of higher
blasts that by DMSO had lost the resistance to apoptotis. potency of myoblasts to fuse into existing myotubes- an
Apoptosis regulating proteins (Bcl-2 family) were indirect proof that quercetin might improve hypertro-
studied in order to find their role in muscle development phying muscle. In contrast, DMSO inhibits myogenesis
[12]. C2C12 myoblasts were Bcl-2 positive (15%), ad- with high efficacity and therefore it should be avoided
ditionally it was thought that the expression of this pro- whenever accelerated muscle development, growth or
tein correlates with early stages of myogenesis. In our regeneration is to be achieved.
experiment, we do provide the evidence that quercetin Acknowledgements
and DMSO modulated and changed Bcl-2 gene expres- This paper is dedicated to the memory of Professor
sion during myogenesis. Therefore, we suppose that Wies aw Barej who supported our efforts to obtain

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 Effect of quercetin and DMSO

funds necessary to complete this study. We thank Mrs. [11] De Vries JHM, Janssen PLTMK, Hollman PCH, Van
Justyna Olczak and Mrs. Teresa Hass for their contri- Staveren WA, Katan MB: Consumption of quercetin
bution to microscopic evaluation of myotube forma- and kaempferol in free-living subjects eating a variety
tion and biochemical analyses. This work was sup- of diets. Cancer Lett 1997; 114: 141-144.
ported by the grant No. 5 PO6K 029 15 from the Pol- [12] Dominov JA, Dunn JJ, Miller JB: Bcl-2 expression
ish State Committee of Sciences. identifies an early stage of myogenesis and pro-
motes clonal expansion of muscle cells. J Cell Biol
Address correspondence to: 1998; 142: 537-544.
Arkadiusz Orzechowski, Department of Physiology, [13] Doumit ME, Cook DR, Merkel RA: Testosterone
Biochemistry, Pharmacology and Toxicology, Faculty up-regulates androgen receptors and decreases dif-
of Veterinary Medicine, Warsaw Agricultural Univer- ferentiation of porcine myogenic satellite cells in
sity, Nowoursynowska 166, 02-787 Warsaw, Poland, vitro. Endocrinol 1996; 137 (4): 1385-1394.
tel. and fax +48 022 847 24 52, Email orzechow- [14] Ferrigno P, Silver PA: Regulated nuclear localiza-
ski@alpha.sggw.waw.pl.. tion of stress-responsive factors: how the nuclear
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