Molecular and Cellular Endocrinology 412 (2015) 116–122

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Molecular and Cellular Endocrinology

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Metformin prevents renal interstitial fibrosis in mice with unilateral

ureteral obstruction
Rita C. Cavaglieri 1, Robert T. Day, Denis Feliers *, Hanna E. Abboud †
Department of Medicine/Nephrology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

A R T I C L E I N F O A B S T R A C T

Article history: Unilateral ureteral obstruction causes important tubulo-interstitial fibrosis in the kidney. Metformin reduces
Received 2 April 2015 fibrosis in mice with diabetic nephropathy. We examined the effects of metformin in a mouse model of
Received in revised form 4 June 2015 unilateral ureteral obstruction (UUO). Expression of inflammation and fibrosis markers was studied by
Accepted 4 June 2015
immunohistochemistry, immunoblot and quantitative real-time polymerase chain reaction. Seven days
Available online 9 June 2015
after UUO, kidneys presented dilated tubules, expansion of the tubulo-interstitial compartment, and sig-
nificant infiltration of inflammatory cells. Macrophage infiltration and inflammation markers expression
Keywords:
were increased in obstructed kidneys and reduced by metformin. Metformin reduced expression of
Metformin
Adenosine monophosphate-activated extracellular matrix proteins and profibrotic factor TGFβ in obstructed kidneys, measured by
kinase immunohistochemistry. Interstitial fibroblast activation was evident in obstructed kidneys and
Kidney ameliorated by metformin. UUO did not affect adenosine monophosphate-activated kinase (AMPK) ac-
Fibrosis tivity, but metformin activated AMPK. Our results show that metformin prevents or slows down the onset
Inflammation of renal inflammation and fibrosis in mice with UUO, an effect that could be mediated by activation of
AMPK.
© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction hemodynamic and metabolic changes, leading to tubular injury and

Obstructive nephropathy can occur in both children and adults.
In adults, renal function usually recovers after elimination of the
obstruction, but in children obstruction can have long term con-
sequences. Fetal and neonatal obstruction result in reduced nephron
number, with the magnitude of reduction being dependent on the
severity and duration of obstruction (Chevalier et al., 2009).
Obstruction is accompanied by changes in kidney structure, in-
flammation and tubulointerstitial fibrosis, leading to decrease and
eventually loss of renal function if the obstruction persists. Inter-
stitial fibrosis is a major prognostic indicator of end-stage renal
disease (Eddy, 2000). Inhibition of interstitial fibrosis is therefore
critical to preserve kidney function (Eddy, 2005). Unilateral ure-
teral obstruction (UUO) is an experimental model for tubulo-
interstitial fibrosis (Chevalier et al., 2009). UUO causes renal
* Corresponding author. Department of Medicine/Nephrology, MC 7882, The
University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San
Antonio, TX 78229, USA. Tel.: +1 210 567 6140; fax: +1 210 567 4712.
E-mail address: feliers@uthscsa.edu (D. Feliers).
1 Present address: Laboratório de Investigação Médica LIM-29, Universidade de
Sao Paulo, Av. Dr. Arnaldo, 455 – 4° andar – sala 4304 –, Cerqueira César, São Paulo,
SP, Brazil.
†
This manuscript is dedicated to the memory of our dear friend and colleague
Hanna E. Abboud who passed away shortly before the completion of the manuscript.
renal inflammation, characterized by macrophage infiltration. Ac-
tivation of interstitial fibroblasts causes their differentiation into
myofibroblasts, which contribute to accumulation of the extracel-
lular matrix proteins, such as fibronection, collagens I and II and
lead eventually to renal fibrosis (for a review, see Chevalier et al.,
2009). Upon recruitment and activation, macrophages produce
various pro-inflammatory cytokines, such as TNFα and TGFβ which
in turn promote expression of adhesion molecules, such as vascu-
lar cell adhesion molecule 1 (VCAM1), and contribute to further
recruitment of circulating inflammatory cells (see Chevalier et al.,
2010 for a review).
Metformin is a biguanide compound that has been used as
an oral anti diabetic drug for over 50 years. It is the first-line drug
of choice for the treatment of type 2 diabetes, in particular, in
overweight and obese people and those with normal kidney func-
tion. Metformin has pleiotropic actions, including inhibition of the
mitochondrial respiratory chain complex I (El-Mir et al., 2000) and
activation of the adenosine monophosphate-activated kinase (AMPK)
(Zhou et al., 2001). In addition to its glycemia-lowering effects,
metformin has been shown to reduce inflammation and fibrosis in
a model of non-alcoholic steato-hepatitis (Kita et al., 2012). However,
the effect of metformin on inflammation and fibrosis in the kidney
has not been studied.
In this study, we studied the effect of treatment with metformin,
initiated 1 day before surgery, on kidney structure, inflammation
and fibrosis in mice with unilateral ureteral obstruction.

http://dx.doi.org/10.1016/j.mce.2015.06.006
0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.
 R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122
2. Materials and methods
2.1. Mouse model of unilateral ureteral obstruction (UUO)
Five- to 7-month-old adult male C57Bl/6 mice, weighting 25–35 g,
were anesthetized with isoflurane/oxygen and UUO was per-
formed using 4-0 silk in the midline abdominal incision and sham-
operated mice had their ureters exposed and manipulated but not
ligated (Cachat et al., 2003). After 7 or 14 days of obstruction, animals
were sacrificed by cervical dislocation and kidneys were decapsu-
lated and weighed. One piece of a kidney was removed for histologic
and immunohistochemical studies and the remaining was frozen
in liquid nitrogen and stored at −80 °C for protein and RNA extrac-
tion. Three groups were used: sham (n = 9), sham-operated mice
receiving vehicle (water) by gavage, UUO (n = 9), mice subjected to
unilateral ureter obstruction model and received vehicle by gavage;
and UUO + metformin (n = 10), UUO mice receiving metformin by
gavage (200 mg/kg/day). All experiments were approved by the Uni-
versity of Texas Health Science Center at San Antonio Institutional
Animal Care and Use Committee.
2.2. Renal histology and Sirius Red staining
Briefly, kidneys were fixed in 10% formaldehyde and embed-
ded in paraffin, and 4-μm sections were cut. Staining with Periodic
Acid Schiff (PAS) and Masson’s Trichrome were performed by the
Cancer Therapy and Research Center at the University of Texas Health
Science Center San Antonio. Images were obtained with an Olympus
AX70 microscope using the DP image acquisition software.
2.3. Immunohistochemistry
Immunohistochemistry was performed as described by Day et al.
(2013).
2.3.1. Fibronectin staining
Antigen retrieval from paraffin-embedded sections (4 μm) was
performed in citrate buffer at 100 °C for 6 minutes, slides were
quenched in 3% hydrogen peroxide for 6 minutes. After blocking with
Background Sniper for 20 minutes, slides were incubated with the
primary antibody (listed in Supplementary Fig. S1) overnight at 4 °C
in a humidified chamber. After rinsing, slides were incubated with
goat anti-rabbit polymer-horseradish peroxidase (Biocare, Concord,
CA) for 20 minutes at room temperature. Immunoreactivity was vi-
sualized with 3-3′-diaminobenzidine (DAB, Biocare, CA).
2.3.2. F4/80, α-SMA and E-cadherin staining
Antigen retrieval from paraffin-embedded sections (4 μm) was
performed in citrate buffer at 100 °C for 6 minutes. Primary anti-
bodies (listed in Supplementary Fig. S1) were incubated overnight
at 4 °C in a humidified chamber. After rinsing, slides were incu-
bated with biotinylated secondary antibody for 20 minutes at room
temperature. Streptavidin–peroxidase complex (LASB®+ System-
AP, DAKO) was used for amplification and immunoreactivity was
visualized with Fast (DAKO).
2.4. Morphometric analysis
Measure of tubular lumen area was performed on slides stained
with E-cadherin. Images were imported in ImageJ64 and the lumen
contour was traced using the free hand tool. Perimeter and surface
area of the trace were determined by the software. Both perime-
ter and surface area show similar results, and we chose to present
only the lumen surface area data, expressed in percentage of lumen
area in sham-operated mice. We measured at least 15 tubules in
3–4 different mice for each group.
117
2.5. Reverse transcription and quantitative real-time polymerase
chain reaction (RT-qPCR)
RNA was extracted from kidney cortex (~50 μg of tissue) using
Trizol (Invitrogen), treated with nuclease I and used for reverse tran-
scription (RT) using iScript RT SuperMix (BioRad). The resulting
cDNAs were used for quantitative PCR using the RT2 qPCR Master
Mix and primers listed in Supplementary Fig. S2. The qPCR was run
in a MasterCycler RealPlex4 (Eppendorf). Quantitation of the mRNAs
was performed using the 2−ΔΔCt method using glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) as a housekeeping gene.
2.6. Immunoblot analysis
Slices of kidneys were homogenized in lysis buffer (Invitrogen,
#FNN-0071) supplemented with protease inhibitor mix (Sigma,
P-2714), 1mM PMSF, and 5 mM orthovanadate. Protein concentra-
tion was measured and 10–20 μg of whole-cell lysates was separated
on SDS–PAGE, transferred to nitrocellulose membranes and probed
overnight at 4 °C with the antibodies listed in Supplementary Fig. S1.
IRDye800- or IRDye700-coupled secondary antibodies were used
for detection using Odyssey Infrared Imaging System (LiCor Bio-
sciences, Lincoln, NE).
2.7. AMPK activity
Briefly, AMPKα was immunoprecipitated from kidney
homogenates using a specific antibody. Immunocomplexes were col-
lected by centrifugation after incubation with protein A/G-agarose
beads. After washes in lysis buffer and washes in PBS, the beads were
resuspended in kinase buffer consisting of 50 mM HEPES (pH 7.4),
10 mM MgCl2, 5% glycerol, 1 mM DTT, 1 mM orthovanadate and 0.05
% Triton X100. Reaction was initiated by addition of 50 μM ATP, 1 μg
SAMS peptide and 10 μCi γ[32P]-ATP and lasted 10 min. An aliquot
of the reaction mixture was spotted on P81 paper disks which were
then washed three times in 0.5% phosphoric acid and once in acetone.
Radioactivity incorporated in the SAMS peptide was counted in a
Beckman 6000IC scintillation counter. Antibodies used are listed in
Supplementary Fig. S1.
2.8. Statistics
Data were expressed as mean ± standard error of the mean (SEM)
and analyzed by analysis of variance (ANOVA) for comparison among
multiple groups using the Tukey post-test analysis for compari-
son, using the GraphPad Prism 5.0 software. Values of p < 0.05 (after
multiple comparison adjustment as needed) were considered
significant.
3. Results
3.1. Renal morphology
Unilateral ureter obstruction was performed in 5- to 7-month-
old mice. Kidney morphology was studied after 1 week of
obstruction. Renal histology was studied after Periodic Acid Schiff
staining of paraffin-embedded kidney sections (Fig. 1A). Ob-
structed kidneys displayed dilated tubules with areas of infiltrating
cells (arrow), suggesting inflammation. Glomeruli were histologi-
cally normal. Metformin treatment significantly reduced the number
of infiltrating cells in obstructed kidneys. E-cadherin is a trans-
membrane protein involved in cell–cell contact and expressed in
epithelial cells (Okada, 1988). In sham-operated kidneys, E-cadherin
was highly expressed in some tubules, and its expression was sig-
nificantly lower in obstructed kidneys (Fig. 1B), indicating loss of
tubular structure. Decrease of E-cadherin expression was attenuated
118 R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122

Fig. 1. Effect of metformin on renal histology and extracellular matrix expansion. (A) Renal histology was studied by Periodic Acid Schiff staining on section of paraffin-
embedded kidneys. 200× magnification. (B) Extracellular matrix expansion was studied by Masson’s Trichrome staining. 100× magnification. N = 4 mice per group.

Fig. 2. Effect of metformin on inflammation in obstructed kidneys. (A) F4/80-positive cells were detected by immunohistochemistry on sections of paraffin-embedded kidneys.
Positive cells stain brown. 200× magnification. N = 4 mice per group. (B) TNFα and VCAM1 expression was measured by immunoblot on homogenates of the left kidney
from sham-operated, UUO and UUO mice treated with metformin. The lower panels show the results of densitometric analysis of the immunoblots. TNFα mRNA (C) and
VCAM1 mRNA (D) expression was measured by RT-qPCR and normalized using GAPDH as a housekeeping gene. N = 6-9 mice per group. *p < 0.05, **p < 0.01 and ***p < 0.001
vs sham by ANOVA, ##p < 0.01 vs UUO by ANOVA.
 R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122 119

by treatment with metformin in obstructed kidneys (Fig. 1B). Next,
we measured the tubular lumen area by morphometry. We found
that the lumen area increased about 5-fold in mice with UUO.
Metformin treatment tended to decrease the lumen surface, but the
difference did not reach statistical significance. These data show that
metformin slows down infiltration of circulating cells and tubular
damage, with a minimal effect on tubular dilatation in obstructed
kidneys.
3.2. Renal inflammation
prevents the increase in TNFα gene transcription, but not a post-
transcriptional regulation of TNFα protein in obstructed kidneys.
Infiltration of inflammatory cells in the kidney requires coordi-
nated expression of several pro-inflammatory molecules, such as
VCAM1 which allows adhesion to the endothelium and infiltra-
tion into the renal parenchyma. VCAM1 expression was increased
at the protein (Fig. 2B) and mRNA level (Fig. 2D) in obstructed
kidneys. Metformin attenuated the increase in VCAM1 expression
in obstructed kidneys, but both mRNA and protein were still higher
in kidneys from mice UUO-metformin that in sham-operated mice.

The PAS staining detected inflammatory cells inside the paren-
chyma of obstructed kidneys. To assess whether these cells contain
macrophages, staining for F4/80 was performed. Fig. 2A shows that
few F4/80 positive cells were present in kidneys from sham-
operated mice. The number of F4/80 positive cells was significantly
increased in obstructed kidneys, mostly in the tubulo-interstitial
space. Treatment with metformin prevented the increase of F4/80
positive cells in obstructed kidneys. These data suggested that
metformin slows down macrophage infiltration in obstructed
kidneys.
TNFα plays an important role in renal inflammation in several
models of kidney injury, including UUO (Guo et al., 1999). TNFα ex-
pression was increased at the protein (Fig. 2B) and the mRNA level
(Fig. 2C) in kidneys from mice with UUO compared to sham-
operated mice. Metformin treatment prevented increased TNFα
protein expression, which remained higher than that in sham-
operated mice. TNFα mRNA levels were completely normalized by
metformin treatment (Fig. 2C). These data suggest that metformin
3.3. Renal fibrosis
Unilateral ureteral obstruction causes progressive accumula-
tion of extracellular matrix in the kidney, leading to tubulo-
interstitial fibrosis. Masson’s Trichrome stain allows detection of
extracellular matrix in tissues, as blue staining. Fig. 3A shows that
obstructed kidneys have significant tubulo-interstitial and glomeru-
lar accumulation of extracellular matrix. Metformin treatment
reduced accumulation of extracellular matrix in obstructed kidneys
(Fig. 3A).
Collagens and fibronectin are two major constituents of extra-
cellular matrix in the kidney. Fibronectin deposition was assessed
by immunohistochemistry: basal staining of fibronectin in the glom-
eruli and the tubulo-interstitial compartment in kidneys from sham-
operated mice was significantly increased in obstructed kidneys
(Fig. 3B). Treatment with metformin reduced accumulation of
fibronectin deposition in obstructed kidneys. Fibronectin expres-
sion was quantified by immunoblot (Fig. 3C) and RT-qPCR (Fig. 3D).

Fig. 3. Effect of metformin on collagen expression in obstructed kidneys. Collagen deposition was measured by Sirius red staining under direct light (A) or indirect light
(B). 100× magnification. N = 4 mice per group. Expression of collagen Iα2 mRNA (C) and collagen IIIα1 mRNA (D) was measured by RT-qPCR and normalized to GAPDH mRNA.
N = 6–9 mice per group. **p < 0.01 and ***p < 0.001 vs sham by ANOVA, #p < 0.01 vs UUO by ANOVA.
120 R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122

Fig. 4. Effect of metformin on fibronectin expression in obstructed kidneys. (A) Fibronectin was detected by immunohistochemistry in sections of paraffin-embedded kidneys.
Positive staining is brown. 100× magnification. N = 4 mice per group. (B) Fibronectin (FN) protein expression was measured by immunoblot on homogenates of the left kidney
from sham-operated, UUO and UUO mice treated with metformin. The lower panels show the results of densitometric analysis of the immunoblots. (C) FN mRNA expres-
sion was measured by RT-qPCR and normalized using GAPDH as a housekeeping gene. N = 6–9 mice per group. **p < 0.01 and ***p < 0.001 vs sham by ANOVA, #p < 0.05 vs
UUO by ANOVA.

Fibronectin protein and mRNA were upregulated in obstructed
kidneys, and treatment with metformin reduce attenuated the in-
creased fibronectin expression in obstructed kidneys.
Collagen deposition was assessed by Sirius Red staining using
direct light, which allows detection of collagens I, III and IV (Fig. 4A),
and polarized light, which allows detection of cross-linked colla-
gens I and III (Fig. 4A). Collagen deposition was increased around
the glomeruli and in the tubulo-interstitial compartment of ob-
structed kidneys. No increase staining inside the glomeruli was
observed. Treatment with metformin attenuated deposition of col-
lagens in obstructed kidneys. Expression of collagen Iα2 (Fig. 4B)
and IIIα1 mRNA (Fig. 4C), measured by RT-qPCR, was increased in
obstructed kidneys and normalized after treatment with metformin.
Transforming Growth Factor β (TGFβ) is an important regula-
tor of fibrosis in several models of kidney injury (Kaneto et al., 1993;
Sato et al., 2003), including the UUO. Its expression was quanti-
fied by immunoblot (Fig. 5A) and RT-qPCR (Fig. 5B). TGFβ protein
and mRNA were upregulated in obstructed kidneys compared to
sham-operated kidneys. Treatment with metformin prevented in-
creased expression of TGFβ protein and mRNA.
Activation of interstitial fibroblasts plays a role in the develop-
ment of tubulo-interstitial fibrosis in mice with UUO (Chevalier,
1999; Grande and López-Novoa, 2009). Fibroblast activation is ac-
companied by upregulation of α-smooth muscle actin (α-SMA)
(Grande and López-Novoa, 2009). In kidneys from sham-operated
mice, α-SMA was localized mainly in the blood vessels, and around
the glomeruli some tubules (Fig. 5C). In obstructed kidneys, α-SMA
was significantly increased in the tubulo-interstitial compart-
ment, and treatment with metformin prevented this increase. The
expression of α-SMA was quantified by immunoblot. Fig. 5D shows
that α-SMA was increased 3.7-fold in obstructed kidneys com-
pared to sham-operated kidneys. Metformin reduced the increase
in α-SMA expression in obstructed kidneys.
Together, these data show that metformin prevents the devel-
opment of renal fibrosis in obstructed kidneys, most likely through
inhibition of TGFβ expression.
3.4. Adenosine monophosphate-activated kinase (AMPK)
Metformin is an indirect activator of AMPK (Viollet et al., 2012).
We measured AMPKα activation by in vitro kinase assay using the
SAMS peptide as a substrate (Fig. 6). Renal AMPKα activity was un-
changed in obstructed kidneys, but treatment with metformin
resulted in a significant increase in AMPKα activity.
4. Discussion
Our study shows for the first time that metformin treatment, ini-
tiated 1 day before surgery, attenuates development of renal
inflammation and fibrosis but not of renal structural changes in mice
with unilateral ureteral obstruction. The latter is a consequence of
urine reflux into the kidney due to the obstruction, and is logical-
ly not affected by metformin. The former complications are due to
the release of soluble factors by the injured kidney tissue. Our data
suggest that this is antagonized by metformin. This is the case for
TNFα, which is involved in inflammation and TGFβ, involved in
fibrosis.
 R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122 121

Fig. 5. Effect of metformin on TGFβ and α-SMA expression in obstructed kidneys. (A) TGFβ protein expression was measured by immunoblot on left kidney homogenates
from the indicated groups of mice. The lower panels show the results of densitometric analysis of the immunoblots. (B) TGFβ mRNA expression was measured by RT-qPCR
and normalized to GAPDH. N = 6–9 mice per group. (C) α-SMA was detected by immunohistochemistry on sections of paraffin-embedded kidneys. Positive staining is brown.
100× magnification. N = 4 mice per group. (D) α-SMA protein expression was measured by immunoblot on left kidney homogenates. The lower panels show the results of
densitometric analysis of the immunoblots. The lower panel shows the results of densitometric analysis of the immunoblots. N = 6–9 mice per group. **p < 0.01 and ***p < 0.001
vs sham by ANOVA, #p < 0.05 vs UUO by ANOVA.

(Kita et al., 2012), and reduced inflammation in the airways of asth-

Fig. 6. Effect of metformin on AMPK activity in obstructed kidneys. AMPK activity
was measured by in vitro kinase assay using SAMS peptide as a substrate, after im-
munoprecipitation of AMPKα using a specific antibody. N = 6 mice per group. **p < 0.01
and ***p < 0.001 vs sham by ANOVA, #p < 0.05 vs UUO by ANOVA.
Metformin has been shown to have anti-inflammatory and anti-
fibrotic effects in other tissues. The anti-inflammatory effects of
metformin have been demonstrated in vivo in the liver and lung.
Metformin reduced expression of pro-inflammatory and pro-
fibrotic genes in a mouse model of non-alcoholic hepatic steatosis
matic mice (Park et al., 2012). The anti-inflammatory effect of
metformin could be due to inhibition of the NF-κB pathway, as evi-
denced in cultured cancer cells (Hirsch et al., 2013), vascular cells
(Isoda et al., 2006), monocytes (Arai et al., 2010) and macrophages
(Hyun et al., 2013). The anti-fibrotic effect of metformin has been
characterized mostly in the cardiovascular system. Metformin
reduced expression of pro-fibrotic genes in hearts of mice with pres-
sure overload (Xiao et al., 2010), in hearts of obese rats (Burlá et al.,
2013), and in hearts from spontaneously hypertensive rats (Cittadini
et al., 2012). The mechanism is likely through antagonism of TGFβ
signaling, as demonstrated in cultured cardiac fibroblasts (Xiao et al.,
2010) and canine kidney epithelial cells (Cufí et al., 2010). Our study
shows for the first time that metformin exerts similar anti-
inflammatory and anti-fibrotic effects in the kidney from mice with
UUO.
Our data show that amelioration of inflammation and fibrosis
in kidneys from metformin-treated mice was accompanied by in-
creased activity of AMPK. Metformin has been shown to activate
AMPK in several organs (Viollet et al., 2012), and although there are
instances when the effects of metformin are independent of AMPK
(Ben Sahra et al., 2011; Kalender et al., 2010), activation of AMPK
is widely believed to mediate the effects of metformin (Viollet et al.,
2012). It is therefore likely that metformin attenuates inflamma-
tion and fibrosis in obstructed kidneys through activation of AMPK.
In support of this hypothesis, it has been shown that pharmaco-
logical activation of AMPK reduces fibrosis in rats with kidney
ablation and infarction (Satriano et al., 2013). Our data show that
AMPK activity is unchanged in obstructed kidneys, suggesting that
122 R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122
inflammation and fibrosis in these kidneys are due to an inhibi-
tion of AMPK activity, in contrast with diabetic nephropathy. In
kidneys from rats with early diabetes, AMPK activity is decreased
and pharmacological activation of AMPK reduces extracellular matrix
protein expression (Lee et al., 2007).
Acknowledgements
This study was supported by the JDRF (1-2010-141) to DF and
the NIDDK (DK-R01-078971) and a VA Merit Review to HEA. Peri-
odic Acid Schiff and Sirius Red staining were performed by the Cancer
Therapy and Research Center at the University of Texas Health
Science Center San Antonio, through the NCI Cancer Center Support
Grant (2 P30 CA054174-17).
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.mce.2015.06.006.
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