On the other hand, activation of PPARc has been shown to inhibit the proliferation of pulmo-nary vascular smooth muscle cells [1,4] and the devel-opment of pulmonary hypertension in anim
Trang 1Heme oxygenase-1 ⁄p21 mediates peroxisome
proliferator-activated receptor-c signaling inhibition of
proliferation of rat pulmonary artery smooth muscle cells Manxiang Li1, Zongfang Li2, Xiuzhen Sun1, Lan Yang3, Ping Fang1, Yun Liu1, Wei Li1, Jing Xu1, Jiamei Lu1, Minxing Xie1 and Dexin Zhang1
1 Department of Respiratory Medicine, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, China
2 Department of General Surgery, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, China
3 Department of Respiratory Medicine, The First Affiliated Hospital of Medical College, Xi’an Jiaotong University, China
Introduction
Peroxisome proliferator-activated receptors (PPARs)
are a group of ligand-activated transcription factors
belonging to the nuclear receptor superfamily PPARs
form heterodimers with retinoid X receptors, binding
to specific PPAR-responsive elements and governing
the expression of relevant genes [1] Three subclasses
of PPARs have been identified: PPARa, PPARb⁄ d,
and PPARc [1] PPARc is expressed predominantly in
adipocytes, activated macrophages, vascular smooth muscle cells, and vascular endothelial cells [2] PPARc
is activated by several natural ligands, such as 15-deoxy-D12,14-prostaglandin J2, 9-hydroxyoctade-cadienoic acid, 3-hydroxyoctade9-hydroxyoctade-cadienoic acid, 12-hy-droxyeicosatetaenoic acid, 15-hy12-hy-droxyeicosatetaenoic acid, and nitro lipids [3] It is also activated by syn-thetic ligands such as thiazolidinediones, e.g troglitazone
Keywords
heme oxygenase-1 (HO-1); p21 WAF1 ;
proliferator-activated receptor-c (PPARc);
pulmonary artery smooth muscle cells;
rosiglitazone
Correspondence
M Li or Z Li, The Second Affiliated Hospital
of Medical College, Xi’an Jiaotong
University, No 157, West 5th Road, Xi’an,
Shaanxi, China 710004
Fax: +86 29 87679463
Tel: +86 29 85520128
E-mail: manxiangli@hotmail.com or
lzf2568@mail.xjtu.edu.cn
(Received 13 November 2009, revised
22 December 2009, accepted 15 January
2010)
doi:10.1111/j.1742-4658.2010.07581.x
Activation of peroxisome proliferator-activated receptor (PPAR)-c sup-presses proliferation of rat pulmonary artery smooth muscle cells (PASMCs), and therefore ameliorates the development of pulmonary hypertension in animal models However, the molecular mechanisms under-lying this effect remain largely unknown This study addressed this issue The PPARc agonist rosiglitazone dose-dependently stimulated heme oxygenase (HO)-1 expression in PASMCs, 5 lm rosiglitazone inducing a 12.1-fold increase in the HO-1 protein level Cells pre-exposed to rosiglitaz-one showed a dose-dependent reduction in proliferation in response to serotonin; this was abolished by pretransfection of cells with sequence-specific small interfering RNA against HO-1 In addition, rosiglitazone stimulated p21WAF1 expression in PASMCs, a 2.34-fold increase in the p21WAF1 protein level being achieved with 5 lm rosiglitazone; again, this effect was blocked by knockdown of HO-1 Like loss of HO-1, loss of p21WAF1 through siRNA transfection also reversed the inhibitory effect of rosiglitazone on PASMC proliferation triggered by serotonin Taken together, our findings suggest that activation of PPARc induces HO-1 expression, and that this in turn stimulates p21WAF1expression to suppress PASMC proliferation Our study also indicates that rosiglitazone, a medi-cine widely used in the treatment of type 2 diabetes mellitus, has potential benefits for patients with pulmonary hypertension
Abbreviations
5-HT, 5-hydroxytryptamine; CDK, cyclin-dependent kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HO, heme oxygenase; PASMC, pulmonary artery smooth muscle cell; PPAR, peroxisome proliferator-activated receptor; siRNA, small interfering RNA.
Trang 2and rosiglitazone, which have been commonly used in
the treatment of type 2 diabetes mellitus [2,3] Reduced
expression of PPARc has been recently reported to be
associated with the development of pulmonary
hyper-tension [4,5] On the other hand, activation of PPARc
has been shown to inhibit the proliferation of
pulmo-nary vascular smooth muscle cells [1,4] and the
devel-opment of pulmonary hypertension in animal models
[1,6] However, the mechanisms by which activation of
PPARc inhibits the proliferation of pulmonary
vascu-lar smooth muscle cells, a pivotal point in pulmonary
vascular remodeling and consequent development of
pulmonary hypertension, are still largely unknown
Heme oxygenase (HO) is the rate-limiting enzyme of
heme catabolism Three isoforms of HO have been
identified: HO-1, HO-2, and HO-3 [7] HO-1 is an
inducible isoform of HO, and its induction has been
shown to be cytoprotective [8] HO catalyzes the
breakdown of heme into iron, biliverdin, and carbon
monoxide [9] Both biliverdin and carbon monoxide
have been found to dilate the vasculature and to
inhi-bit the proliferation of vascular smooth muscle cells
[10] Induction of HO-1 by either genetic approaches
or pharmacological intervention has been shown to be
effective in preventing or treating pulmonary
hyperten-sion in animal models [11,12] A recent study has
sug-gested that activation of PPARc induces expression of
HO-1 in human umbilical vein endothelial cells and
human umbilical artery or vein smooth muscle cells
[13] However, it is still unknown whether activation of
PPARc also stimulates HO-1 expression in pulmonary
artery smooth muscle cells (PASMCs) (vascular
smooth muscle cells showing some differences from
systemic vascular smooth muscle cells) If so, whether
and how HO-1 induction further inhibits proliferation
of PASMCs are still unclear, especially stimulated with
several mitogenic agonists involved in the pathogenesis
of pulmonary hypertension, such as serotonin
[5-hydroxytryptamine (5-HT)] and endothelin-1 [14,15]
Vascular smooth muscle cells are normally
quies-cent, and remain in the G1⁄ G0 phase of the cell cycle
However, upon stimulation, cells exit the G1⁄ G0phase
and start to divide [16] Cell cycle progression is
precisely controlled by the activity of a series of
cyclin-dependent kinases (CDKs), which are activated by
cyclin binding and negatively regulated by CDK
inhibitors P21WAF1 is one of several important CDK
inhibitors [17] We hypothesized that activation of
PPARc could induce the expression of HO-1, and that
this in turn could upregulate the expression of
p21WAF1, leading to suppression of PASMC
prolife-ration To test our hypothesis, we isolated and
cultured primary PASMCs, and determined the
impact of activation of PPARc on the expression of HO-1 and p21WAF1 We also explored whether these responses modulate cell proliferation induced by 5-HT
Results
Effect of PPARc agonist on HO-1 expression Activation of PPARc by rosiglitazone has been shown
to induce the expression of HO-1 in several types of mammalian cells, including non-PASMCs; however, this effect has not been reported in PASMCs to date To examine this effect in pulmonary vascular smooth mus-cle cells, we treated PASMCs with various concentra-tions of rosiglitazone for 24 h, and analyzed the expression of HO-1 using western blotting As shown in Fig 1, cells treated with rosiglitazone displayed a dose-dependent increase in HO-1 expression As compared with control cells, 5 lm rosiglitazone caused a 12.1-fold increase in protein expression of HO-1 (P < 0.01), sug-gesting that activation of PPARc specifically and effec-tively mediates HO-1 induction in PASMCs
Role of HO-1 in PPARc agonist suppression of proliferation of PASMCs
HO-1 has been found to be highly effective against pulmonary hypertension, through vasodilating, inhibit-ing the inflammatory response, and suppressinhibit-ing the proliferation of PASMCs At the same time, activation
HO-1
GAPDH
Con
Rosiglitazone
0 5 10 15 20
**
**
**
Fig 1 The PPARc agonist rosiglitazone induces HO-1 expression Primary cultured PASMCs were stimulated with different concen-trations of rosiglitazone for 24 h The expression of HO-1 was determined using immune blotting GAPDH was used as loading control Representative western blotting and quantification of bands are shown (n = 3 in each group) **P < 0.01 versus control (Con).
Trang 3of PPARc has also been shown to inhibit the
prolifera-tion of PASMCs and thus to ameliorate the
develop-ment of pulmonary hypertension It is therefore
interesting to know whether induction of HO-1
medi-ates the protective effect of PPARc against PASMC
proliferation To test this, we applied serotonin to
stimulate PASMC proliferation, and then examined
whether knockdown of HO-1 by small interfering
RNA (siRNA) attenuated the effect of PPARc agonist
on cell proliferation Figure 2A shows that PASMCs
stimulated with 5-HT (1 lm for 24 h) exhibited
4.21-fold increase in DNA synthesis as assessed by [3
H]thy-midine incorporation assay (P < 0.01 as compared
with control), and pretreatment of cells with the
PPARc agonist rosiglitazone for 12 h
dose-depen-dently suppressed 5-HT-induced cell proliferation At
5 lm, rosiglitazone fully inhibited 5-HT-triggered
DNA synthesis in cells (Fig 2A) Figure 2B shows that
sequence-specific HO-1 siRNA transfection for 72 h
reduced basal HO-1 expression by 91% (P < 0.01
ver-sus control), whereas nontargeting siRNA transfection
did not change the HO-1 level More importantly, we
found that prior HO-1 knockdown by siRNA
abol-ished the inhibitory effect of rosiglitazone on the
pro-liferation of PASMCs induced by 5-HT (Fig 2C),
whereas HO-1 knockdown alone did not impact on
basal or 5-HT-stimulated DNA synthesis in cells Our
study indicates that induction of HO-1 mediates the
suppressive effect of PPARc agonist on PASMC
proliferation
Role of p21WAF1in HO-1-mediated suppression of
proliferation of PASMCs
Recent studies have revealed that an antiproliferative
effect of HO-1 on non-PASMC pulmonary vascular
smooth muscle cells and other types of cells is
associ-ated with upregulation of the CDK inhibitor p21WAF1,
which is involved in negative regulation of cellular
pro-liferation [18,19] We thus determined whether
increased HO-1 expression induced by PPARc agonist
could, in turn, trigger upregulation of p21WAF1,
lead-ing to an increase in its activity against PASMC
prolif-eration stimulated with 5-HT Figure 3 shows that
PASMCs treated with rosiglitazone (5 lm for 24 h)
displayed a 2.34-fold increase in expression of p21WAF1
(P < 0.01 as compared with control), whereas this
increase was dramatically blocked by prior knockdown
of HO-1, suggesting that HO-1 induction caused by
PPARc agonist is apparently involved in the
upregula-tion of p21WAF1 in PASMCs To further confirm this
observation functionally, we examined whether
knock-down of p21WAF1 by siRNA transfection could reverse
the effect of PPARc agonist on suppression of PASMC proliferation We first applied sequence-spe-cific siRNA to knock down expression of p21WAF1 As shown in Fig 4A, transfection of p21WAF1 siRNA for
72 h produced an 82% reduction in p21WAF1 protein
0 100 200 300 400 500
5-HT
0 0 0.5 1.5 5 µ M
0 + + + + 1 µ M
rosiglitazone
**
** #
**##
##
Rosi 5-HT
Rosi 5-HT
HO-1 siRNA HO-1 siRNA 5-HT
HO-1 GAPDH
0 100 200 300 400 500
600
**
##
**
##
** ††
0 0.5 1 1.5
**
Con Non-targeting HO-1 siRNA
siRNA
C
B A
Fig 2 HO-1 mediates the inhibitory effect of the PPARc agonist rosiglitazone (Rosi) on PASMC proliferation (A) Primary cultured PASMCs were stimulated 5-HT (1 l M for 24 h), and this was followed by labeling with [ 3 H]thymidine (1 lCiÆmL)1 for 12 h) Rosiglitazone was added 12 h before stimulation of cells with 5-HT Cells were lysed, and cell-associated radioactivity was measured by liquid scintillation counting Summary data show that rosiglitazone dose-dependently suppressed 5-HT-induced DNA synthesis (n = 4
in each group) (B) Primary cultured PASMCs were transfected with HO-1 sequence-specific siRNA (HO-1 siRNA) or nontargeting con-trol siRNA for 72 h Equal amounts of protein were loaded and probed using specific HO-1 and GAPDH (loading control) antibodies Representative western blotting and quantification of HO-1 bands are shown (C) Prior knockdown of HO-1 by siRNA significantly reversed the inhibitory effect of rosiglitazone on DNA synthesis in 5-HT-treated cells (n = 4 in each group) **P < 0.01 versus control;
#P < 0.05, ##P < 0.01 versus 5-HT-treated cells; P < 0.01 versus rosiglitazone and 5-HT-treated cells.
Trang 4level (P < 0.01 versus control), whereas nontargeting
control siRNA transfection did not change the
p21WAF1 level in cells Next, we investigated the
influ-ence of the loss of p21WAF1 on the effect of PPARc activation on suppression of cell proliferation Fig-ure 4B indicates that knockdown of p21WAF1 by siRNA transfection significantly reversed the inhibitory effect of PPARc agonist on PASMC proliferation induced by 5-HT The DNA synthesis rate was increased again from a 1.4-fold increase over control
in cells treated with PPARc agonist and 5-HT to a 4.04-fold increase over control in cells with p21WAF1 siRNA silencing (despite the presence of PPARc ago-nist and 5-HT), which is similar to that in cells stimu-lated with 5-HT alone or cells treated with the combination of HO-1 siRNA transfection, PPARc agonist, and 5-HT These results suggest that upregu-lation of p21WAF1 by HO-1 mediates the effect
of PPARc agonist in suppression of PASMC prolife-ration
Discussion
In this study, we demonstrate that activation of PPARc by rosiglitazone induces significant HO-1 expression in primary cultured PASMCs, and this sub-sequently upregulates the expression of p21WAF1, lead-ing to inhibition of proliferation of PASMCs stimulated with 5-HT The present study provides a
P21
GAPDH
HO-1 siRNA rosiglitazone Con Rosiglitazone
0
1
2
3
**
##
Fig 3 HO-1 mediates the effect of the PPARc agonist
rosiglitaz-one in upregulating p21 WAF1 expression Primary cultured PASMCs
were treated with rosiglitazone (5 l M ), with or without prior
knock-down of HO-1, for 24 h Expression of p21 WAF1 was determined
using immune blotting GAPDH was used as loading control (Con).
Representative western blotting and quantification of bands are
shown (n = 4 in each group) **P < 0.01 versus control;
##P < 0.01 versus rosiglitazone-treated cells.
P21
GAPDH
0
0.2
0.4
0.6
0.8
1
1.2
**
p21 siRNA Con Non-targeting
siRNA
Con 5-HT Rosiglitazone
5-HT
HO-1 siRNA rosiglitazone 5-HT
p21 siRNA rosiglitazone 5-HT
0
100
200
300
400
500
600
##
** ††
B
A
Fig 4 p21 WAF1 mediates the inhibitory effect of HO-1 on PASMC proliferation (A) Primary cultured PASMCs were transfected with p21 WAF1 sequence-specific siRNA (p21 siRNA) or nontargeting control siRNA for 72 h Equal amounts of protein were loaded and probed using specific p21 WAF1 and GAPDH (loading control) antibodies Representative western blotting and quantification of p21 WAF1 bands are shown (B) Primary cultured PASMCs with
or without prior p21 WAF1 or HO-1 siRNA transfection were stimulated with 5-HT (1 l M for 24 h), and this was followed by labeling with [ 3 H]thymidine (1 lCiÆmL)1for
12 h) Rosiglitazone (5 l M ) was added 12 h before stimulation of cells with 5-HT Cells were lysed, and cell-associated radioactivity was measured by liquid scintillation counting (n = 4 in each group) **P < 0.01 versus control; ##P < 0.01 versus 5-HT-treated cells; P < 0.01 versus cells treated with rosiglitazone and 5-HT.
Trang 5novel molecular mechanism by which PPARc
activa-tion suppresses PASMC proliferaactiva-tion and therefore
ameliorates the development of pulmonary
hyperten-sion It also indicates that rosiglitazone might be useful
in the treatment of pulmonary hypertension
Activation of PPARc by pharmacological ligands
has been shown to exert inflammatory and
anti-proliferative effects on a variety of cell types, and thus
has potential value in the treatment of multiple
diseases [2,20–22] Recent evidence from studies with
animal models indicates that the enhancing activity of
PPARc attenuates the development of pulmonary
hypertension [4,6,23] Further studies suggest that
acti-vation of PPARc confers protection against
pulmo-nary hypertension by suppressing PASMC
proliferation Proliferation of PASMCs is a hallmark
of pathogenesis of pulmonary hypertension [1,4]
How-ever, the mechanisms responsible for inhibition of
PASMC proliferation by activation of PPARc are still
largely unknown Recent studies have suggested that
induction of HO-1 mediates the effect of activation of
PPARc against proliferation of non-PASMCs and
endothelial cells [13] In the present study, we show
that the synthetic PPARc agonist rosiglitazone
dose-dependently inhibited 5-HT-stimulated proliferation of
PASMCs, and that this was accompanied by a
dose-dependent increase in expression of HO-1 Knockdown
of HO-1 abolished the inhibitory effect of PPARc
agonist on PASMC proliferation, suggesting that
induction of HO-1 fully mediates this effect Our study
not only confirms previous findings, but also extends
this notion to the pulmonary system
Mammalian cell proliferation is controlled by a
group of cell cycle protein complexes consisting of two
key regulatory molecules: CDKs and cyclins [17,24]
A CDK molecule is activated by association with a
cyclin, forming a CDK complex CDKs are
constitu-tively expressed in cells, whereas cyclins are synthesized
at specific stages of the cell cycle [25] The expression
of a cyclin is regulated at the transcriptional and
degradation level to influence CDK activity [26] In
addition, CDK activity is modulated by a group of
CDK inhibitors comprising two families of proteins:
inhibitor of kinase 4⁄ alternative reading frame and
CDK inhibitor protein⁄ kinase inhibitor protein The
inhibitor of kinase 4⁄ alternative reading frame family
includes p16INK4a and p14arf, which bind to CDK4
and arrest the cell cycle in the G1phase or prevent p53
degradation, respectively [27,28] The CDK inhibitor
protein⁄ kinase inhibitor protein family includes
p21WAF1, p27Kip1, and p57Kip2 They halt the cell cycle
in the G1 phase by binding to, and inactivating,
cyclin–CDK complexes [29,30] The results of our
study reveal that activation of PPARc increases p21WAF1 expression, and that this effect is significantly blocked by prior knockdown of HO-1 This indicates that PPARc agonist-induced HO-1 expression mediates p21WAF1 upregulation We further confirmed this observation functionally by using p21WAF1 siRNA silencing, when loss of p21WAF1 significantly reversed the inhibitory effect of PPARc agonist on cell proli-feration Our result is consistent with that of Pae [31], showing that curcumin-induced HO-1 expression regu-lates p21 expression in aortic smooth muscle cells The mechanisms underlying HO-1 induction of p21WAF1 expression may be explained by accumulation of iron and carbon monoxide, two key products of HO-1 [32] Pulmonary hypertension and consequent cor pulmo-nale, particularly secondary to chronic obstructive pul-monary disease, are common clinical conditions and some of the major causes of hospitalization and death
in patients with chronic obstructive pulmonary disease [33,34] Increased pulmonary vascular resistance caused
by pulmonary vasoconstriction and vascular remodel-ing (prominent with vascular smooth muscle cell pro-liferation) is the major basis for the development of pulmonary hypertension [35,36] Most drugs currently used in the treatment of pulmonary hypertension are vasodilators; few are aimed effectively against pulmo-nary vascular remodeling [37,38], which is considered
to be a more critical mechanism for chronic pulmonary hypertension [39] Therefore, putative candidates to modulate vascular remodeling have important poten-tial applications in the treatment of pulmonary hyper-tension Rosiglitazone is a wildly used medicine with beneficial effects in the long-term treatment of diabetic mellitus [40] Accumulated clinical experience and the safety record of rosiglitazone suggest that this may be
an important chronic therapeutic approach for human pulmonary hypertensive disease
Experimental procedures
Cell preparation and culture Primary smooth muscle cells from pulmonary arteries were prepared from Sprague–Dawley rats (125–250 g) by the method reported by Golovina et al [41] Isolated arterial rings were incubated in Hanks’ balanced salt solution con-taining 1.5 mgÆmL)1 collagenase II (Worthington, Lake-wood, NJ, USA) for 20 min After incubation, a thin layer
of the adventitia was carefully stripped off with fine forceps, and the endothelium was removed by gently scratching the intima surface with a surgical blade The remaining smooth muscle was then digested with 2.0 mgÆmL)1 collagenase II and 0.5 mgÆmL)1 elastase IV
Trang 6(Sigma, St Louis, MO, USA) for 45 min at 37C The cells
were plated onto 10 cm Petri dishes containing DMEM
(Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine
serum, 100 UÆmL)1 penicillin, and 100 lgÆmL)1
strepto-mycin, and cultured in a 37C ⁄ 5% CO2humidified
incuba-tor Cells were passaged by trypsinization, using 0.05%
trypsin⁄ EDTA (Invitrogen) All experiments were
per-formed using cells between passages 4 and 8 To test the
purity of smooth muscle cells, cells were stained with
4¢,6¢-diamidino-2-phenylindole (Invitrogen) and fluorescein
isothiocyanate-labeled antibody against smooth muscle
a-actin (Sigma), for nucleus and smooth muscle actin,
respectively Fluorescence microscope images indicated that
cells contained more than 93% smooth muscle cells (data
not shown here) Before each experiment, cells were
incubated in 0.5% fetal bovine serum⁄ DMEM for 24 h to
minimize serum-induced effects on the cells 5-HT (Sigma)
was used to stimulate the proliferation of PASMCs
Rosig-litazone (Cayman Chemical Co., Ann Arbor, MI, USA)
was used to stimulate PPARc activation
siRNA transfection
To silence the expression of HO-1 and p21WAF1 protein,
PASMCs were transfected with sequence-specific or
nontar-geting control siRNA (Dharmacon, Lafayette, CO, USA),
using Lipofectamine 2000 reagent (Invitrogen) Briefly, cells
were cultured up to 30–40% confluence, and siRNA and
Lipofectamine were diluted in serum-free DMEM
sepa-rately and incubated for 5 min at room temperature
siRNA was mixed with Lipofectamine and incubated at
room temperature for 20 min Then, the complex of siRNA
and Lipofectamine was added to cells, and culture was
maintained for 72 h at 37C and 5% CO2in a humidified
incubator Cells were transfected for 24 h before the
prepa-ration of the [3H]thymidine incorporation assay The effect
of protein silencing was analyzed using western blot
Immunoblotting
Cells were lysed in 50 mm Tris⁄ HCl (pH 7.4), 1% Nonidet
P-40, 0.1% SDS, 150 mm NaCl, 0.5% sodium
deoxycho-late, 1 mm EDTA, 1 mm phenylmethanesulfonyl fluoride,
1 mm Na3VO4, 1 mm NaF, and proteinase inhibitors
Lysates were centrifuged at 15 700 g at 4C for 15 min,
and the supernatant was collected as total protein The
pro-tein concentration was determined with a bicinchoninic acid
protein assay kit (Pierce, Rockford, IL, USA) Protein was
separated on an SDS⁄ PAGE gel, and transferred to a
Trans-Blot nitrocellulose membrane (Bio-Rad, Hercules,
CA, USA) Monoclonal antibodies against p21WAF1 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
polyclonal antibody against HO-1 (Millipore, Bedford,
MA, USA) were used according to the manufacturer’s
instructions Horseradish peroxidase-conjugated goat
anti-(mouse IgG) and goat anti-(rabbit IgG) were used as sec-ondary antibodies (Sigma) Reactions were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposure to autoradiographic film Signaling was quan-tified from scanned films using scion nih image software (Scion, Frederick, MD, USA)
[3H]Thymidine incorporation assay PASMCs were grown to 50–60% confluence in 24-well plates, and serum starved for 24 h (0.5% fetal bovine serum
in DMEM) before the start of experiments Cells were treated with 1 lm 5-HT or vehicle for 24 h, and this was followed by labeling with [3H]thymidine (1 lCiÆmL)1) for
12 h PPARc agonist was added 12 h before the stimulation
of cells with serotonin After labeling, cells were washed twice with ice-cold NaCl⁄ Piand incubated in 5% trichloro-acetic acid for 30 min at 4C Cell lysates were then washed with ice-cold NaCl⁄ Pi and solubilized by adding 0.5 mL of 0.5 m NaOH⁄ 0.5% SDS Cell-associated radio-activity was measured by liquid scintillation counting
Statistics Values are presented as mean ± standard deviation Data were analyzed using one-way ANOVA with Tukey post hoc test P < 0.05 was considered to represent significant differences between groups
Acknowledgements
This work was supported by the Chinese National Science Foundation (30871116), the Tengfei Talent Project of Xi’an Jiaotong University and the start-up package to M Li from the Second Affiliated Hospital
of Medical College of Xi’an Jiaotong University, PR China
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