Manzanilla
Manzanilla
Manzanilla
Author contributions: K.M., E.G., and A.I.D. designed research; D.A., K.M., A.Y., A.P., and
B.B. performed research; K.K. contributed new reagents/analytic tools; D.A., K.M., E.G., and
A.I.D. analyzed data; and E.G. and A.I.D. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission. D.D. is a guest editor invited by the Editorial
Board.
Freely available online through the PNAS open access option.
1
APPLIED BIOLOGICAL
SCIENCES
Edited by Dean DellaPenna, Michigan State University, East Lansing, MI, and accepted by the Editorial Board April 23, 2013 (received for review
February 27, 2013)
inserts with primers in the phage arms (Table S1) and attaching
indexed adapters to allow multiplexed sequencing (Fig. 1D).
From a total of 8.3 million indexed 35-bp-long reads corresponding to the seven different libraries obtained (original library, Input1, C-E1, A-E1, Input2, C-E2, and A-E2) (Fig. 1C),
64% aligned with ORFs in the human genome (Table S2). To
discover putative apigenin targets, the following criteria to the
identied sequences were applied. (i) Inserts had to be in frame
with the phage capsid, (ii) sequences had to be enriched in
A- compared with C-beads (Fig. S2 A and B), and (iii) reads had
to match one or multiple sequences in the human genome. In
those cases where reads aligned to multiple coding sequences,
weighted counts were used to obtain normalized in frame-aligned
counts per gene models (nICPGs) (Fig. S1B). From a minimum
of 15,568 genes represented in the original phage display cDNA
library, which was established from combining sequence information from the original and selected fractions, 160 genes meeting
these three criteria were identied (Fig. 1E), considered here the
candidate apigenin targets (Table 1).
Simultaneously to PD-Seq, we characterized clones recovered
after the third round of panning by the traditional phage display
approach by performing bacterial infections and individual plaque analysis (Fig. S3 A and B). As is usually the case (25, 26), this
approach yielded a very small number of clones. We analyzed
a total of 16 plaques by PCR. Three of them showed a small
fragment, which on sequencing, proved to be identical to each
other. They corresponded to what we have named the MKET
clone, which we found to be highly enriched in all fractions;
however, it may possibly be an artifact of library preparation
(Fig. S3 BF). The remaining 13 clones had similarly sized
inserts, and sequencing all of them determined that they corresponded to the C-terminal region (residues 264341) (Fig. S3C)
of hnRNPA2, also identied as the top candidate by PD-Seq
(Table 1).
HnRNPA2 and its splice variant hnRNPB1 play fundamental
roles in the progression of tumorigenesis by regulating splicing,
mRNA stability, and mRNA transport (27). Consistent with its
presence in the phage display library used here, hnRNPA2/B1
higher expression has been reported in several human cancers,
including breast (28), and hnRNPA2 expression is recognized as
a marker of glioblastoma and lung cancer (2931).
The hnRNPA2 C-terminal region (hnRNPA2C) contains three
of six YGGG repeats that characterize the proteinprotein interaction glycine-rich domain (hnRNPA2GRD) (Fig. 2B) (32). As a
rst step to determine the specicity of hnRNPA2C for different
avonoids, we performed A-bead pull-down assays of hnRNPA2C
phage (-hnRNPA2C) suspensions in the presence of apigenin or
naringenin. Apigenin, but not naringenin, signicantly competed
the binding of -hnRNPA2C to A-beads (Fig. S4A).
The identication of just hnRNPA2 by conventional phage
display screening underscores one of its main limitations, namely
the retrieval of very few candidates, likely because many bona
de targets fail to be properly amplied or selected through
multiple biopanning rounds (33). The results here presented
show that PD-Seq overcomes this shortcoming, providing an
opportunity for the high-throughput identication of smallmolecule targets.
Apigenin Targets Are Enriched in Three Main Categories. A comparison of the sequence of 160 putative apigenin targets failed
to reveal any obvious common protein domains or stretches
of conserved amino acids. However, analyses based on known
functional annotations and gene ontology (GO) showed that
three main categories (GTPase activation, membrane transport,
and mRNA metabolism/alternative splicing) are highly signicantly (P < 0.01) overrepresented among the identied 160
apigenin targets (Table 1 and Fig. S2). The GTPase activation
functional category contains proteins such as Rho-guanine
nucleotide exchange factor 1 (ARHGEF1) involved in the activation of Rho-GTPases (34), a family of proteins regulating cell
Arango et al.
PNAS PLUS
OH
HO
O2N
H
OH O
+
OH
Br
NH2
Br
OH O
pyridine
K2CO 3
DMF
N
H
OH
+
O
NH2
Ac2O
pyridine
CH3
N
H
N
H
OH
HO
D
T7 phage left arm
cDNA
3X
E2
A-
t2
E2
C-
E1
pu
A-
In
ri-
C
Wash
In
lib
pu
t1
CE1
3X
Elute
Amplify
Bio-pannings
2nd
Elution
ficatio
3rd
Ampli
tion
Amp
Amp
lifica
lifica
tion
1st
Elution
Generation of Illumina
a GAII libraries
Elution
C-E3A-E3
log10(nICPG)
3.0
APPLIED BIOLOGICAL
SCIENCES
Gene name
P value*
GO
ENSG number
Gene name
P value*
GO
ENSG00000122566
ENSG00000125351
ENSG00000121236
ENSG00000109906
ENSG00000143476
ENSG00000109132
ENSG00000197768
ENSG00000168038
ENSG00000116544
ENSG00000105643
ENSG00000137766
ENSG00000214773
ENSG00000213689
ENSG00000187546
ENSG00000076928
ENSG00000170832
ENSG00000134668
ENSG00000140279
ENSG00000137857
ENSG00000169126
ENSG00000103313
ENSG00000182175
ENSG00000008197
ENSG00000090920
ENSG00000159200
ENSG00000203989
ENSG00000131721
ENSG00000204620
ENSG00000058668
ENSG00000080031
ENSG00000186517
ENSG00000179542
ENSG00000107897
ENSG00000117385
ENSG00000035928
ENSG00000181295
ENSG00000119929
ENSG00000154914
ENSG00000116991
ENSG00000160007
ENSG00000103197
ENSG00000196440
ENSG00000074621
ENSG00000139223
ENSG00000107937
ENSG00000086200
ENSG00000118096
ENSG00000033327
ENSG00000092820
ENSG00000145861
ENSG00000149187
ENSG00000139842
ENSG00000141424
ENSG00000181381
ENSG00000166341
ENSG00000109814
ENSG00000153902
ENSG00000115850
ENSG00000107821
ENSG00000056736
ENSG00000187800
ENSG00000212882
ENSG00000178996
HNRNPA2B1
UPF3B
TRIM34
ZBTB16
DTL
PHOX2B
KRT17
ULK4
DLGAP3
ARRDC2
UNC13C
AC112512.6
TREX1
TMEM195
ARHGEF1
USP32
SPOCD1
DUOX2
DUOX1
ARMC4
MEFV
RGMA
TFAP2D
FCGBP
DSCR1
RHOXF2B
RHOXF2
AF196972.1
ATP2B4
PTPRH
ARHGAP30
SLITRK4
ACBD5
LEPRE1
RFC1
AL031289.1
CUTC
USP43
SIPA1L2
GRLF1
TSC2
ARMCX4
SLC24A1
ANP32D
GTPBP4
IPO11
IFT46
GAB2
VIL2
C1QTNF2
CUGBP1
CUL4A
SLC39A6
DDX60L
DCHS1
UGDH
LGI4
LCT
KAZALD1
IL17RB
PEAR1
AL139010.29
SNX18
<1E-300
<1E-300
<1E-300
<1E-300
<1E-300
1.51E-273
3.81E-271
3.89E-261
1.83E-245
1.88E-183
7.04E-133
1.51E-123
6.34E-117
2.30E-110
2.92E-107
2.15E-92
3.14E-89
3.14E-89
3.14E-89
3.98E-89
6.28E-89
8.43E-81
2.16E-78
2.76E-76
1.77E-74
7.37E-70
7.37E-70
1.16E-69
1.11E-55
1.42E-38
4.46E-38
3.76E-37
4.93E-32
2.64E-30
2.52E-29
8.08E-28
2.58E-26
5.17E-26
8.27E-25
6.62E-24
1.32E-23
1.32E-23
3.43E-23
1.06E-22
4.02E-21
2.50E-20
3.09E-20
9.43E-20
1.20E-19
4.34E-19
4.34E-19
2.17E-18
3.77E-18
1.39E-17
2.78E-17
1.11E-16
2.22E-16
8.88E-16
3.55E-15
7.11E-15
5.68E-14
5.68E-14
1.14E-13
c
c, d
c
c
b,c,d
d
d
d
b,d
c
b,d
d
c,d
b,d
a,b,c,d
b,d
c
b
b
d
c
b,c
d
b
c
d
d
d
b,c,d
b,c,d
a,c,d
b
b,c,d
c
c,d
d
d
c,d
a,c,d
a,c
a,b,c,d
d
b,c,d
d
d
d
d
b,c,d
b
b
c
c,d
b,c,d
c
b
d
c
b
c
b,c
b,d
b,d
b,c,d
ENSG00000110975
ENSG00000123178
ENSG00000138442
ENSG00000182077
ENSG00000157193
ENSG00000175471
ENSG00000105245
ENSG00000112273
ENSG00000147454
ENSG00000213380
ENSG00000146950
ENSG00000122034
ENSG00000106484
ENSG00000119812
ENSG00000112701
ENSG00000102452
ENSG00000145934
ENSG00000144354
ENSG00000165185
ENSG00000159784
ENSG00000203943
ENSG00000166845
ENSG00000127951
ENSG00000215811
ENSG00000155984
ENSG00000150938
ENSG00000026036
ENSG00000138735
ENSG00000087087
ENSG00000168495
ENSG00000134490
ENSG00000165125
ENSG00000160293
ENSG00000101096
ENSG00000166471
ENSG00000182261
ENSG00000101220
ENSG00000126768
ENSG00000010704
ENSG00000113448
ENSG00000115290
ENSG00000126803
ENSG00000184650
ENSG00000115275
ENSG00000196792
ENSG00000183783
ENSG00000196381
ENSG00000187621
ENSG00000170734
ENSG00000105613
ENSG00000153944
ENSG00000124260
ENSG00000198216
ENSG00000205334
ENSG00000002016
ENSG00000088756
ENSG00000072182
ENSG00000151693
ENSG00000120251
ENSG00000102858
ENSG00000067057
ENSG00000178952
ENSG00000181036
SYT10
C13orf1
WDR12
PTCHD3
LRP8
MCTP1
NUMBL
HDGFL1
SLC25A37
COG8
SHROOM2
GTF3A
MEST
FAM98A
SENP6
VGCNL1
ODZ2
CDCA7
KIAA1958
FAM131B
SAMD13
C18orf54
FGL2
BTNL10
TMEM185A
CRIM1
TNFRSF6B
PDE5A
SRTT
POLR3D
TMEM241
TRPV6
VAV2
NFATC2
TMEM41B
NLRP10
C20orf27
TIMM17B
HFE
PDE4D
GRB14
HSPA2
ODF4
MOGS
STRN3
KCTD8
ZNF781
TCL6
POLH
MAST1
MSI2
MAGEA10
CACNA1E
AC074091.13
RAD52
ARHGAP28
ACCN4
DDEF2
GRIA2
MGRN1
PFKP
TUFM
FCRL6
2.84E-09
3.73E-09
3.73E-09
3.73E-09
7.45E-09
1.49E-08
7.45E-09
4.88E-04
1.49E-08
1.49E-08
2.98E-08
2.98E-08
1.19E-07
1.19E-07
2.38E-07
4.63E-07
4.77E-07
4.77E-07
4.77E-07
4.77E-07
3.81E-06
3.81E-06
3.81E-06
3.81E-06
7.63E-06
7.63E-06
1.53E-05
3.05E-05
3.05E-05
3.05E-05
3.05E-05
3.05E-05
3.05E-05
3.05E-05
3.05E-05
4.08E-05
6.10E-05
6.10E-05
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
2.44E-04
4.88E-04
9.77E-04
9.77E-04
9.77E-04
9.77E-04
9.77E-04
1.95E-03
1.95E-03
1.95E-03
1.95E-03
1.95E-03
1.95E-03
b
c
d
b
b,c,d
b,c
d
d
b,c,d
b,d
b,d
c,d
b,c
d
c,d
b,c
b,d
c
c,d
d
c
c
d
b
b
b,d
c,d
c,d
d
d
b,c
b,c,d
a,c,d
c,d
b,c,d
d
d
b,d
b,c,d
b,c,d
b,d
b
b
b
b,c,d
d
c
d
c,d
b,d
c,d
d
b,c,d
d
c,d
a,c,d
b,c,d
a,b,c
b,c,d
c,d
d
d
b,c,d
E2156 | www.pnas.org/cgi/doi/10.1073/pnas.1303726110
Arango et al.
PNAS PLUS
ENSG number
Gene name
P value*
GO
ENSG number
Gene name
P value*
GO
ENSG00000085433
ENSG00000151303
ENSG00000184935
ENSG00000116176
ENSG00000176009
ENSG00000142319
ENSG00000101489
ENSG00000008311
ENSG00000185499
ENSG00000020256
ENSG00000107262
ENSG00000166948
ENSG00000063761
ENSG00000011451
ENSG00000100441
ENSG00000114859
ENSG00000110057
WDR47
AGAP11
AC090510.4
TPSG1
ASCL3
SLC6A3
BRUNOL4
AASS
MUC1
ZFP64
BAG1
TGM6
ADCK1
WIZ
KHNYN
CLCN2
UNC93B1
2.12E-13
2.27E-13
9.09E-13
9.09E-13
9.09E-13
9.09E-13
1.82E-12
1.82E-12
3.64E-12
1.64E-11
5.82E-11
1.16E-10
1.16E-10
4.66E-10
4.66E-10
4.66E-10
9.31E-10
c
a
d
b
b
b,d
c
b
b,c,d
c,d
c
c
c,d
c,d
d
b,c,d
b,d
ENSG00000177034
ENSG00000171160
ENSG00000092847
ENSG00000162981
ENSG00000185686
ENSG00000152894
ENSG00000124279
ENSG00000112112
ENSG00000067829
ENSG00000151989
ENSG00000157551
ENSG00000174307
ENSG00000117984
ENSG00000157093
ENSG00000176700
ENSG00000145242
ENSG00000155975
MTX3
MORN4
EIF2C1
FAM84A
PRAME
PTPRK
FASTKD3
COL11A2
IDH3G
C2orf21
KCNJ15
PHLDA3
CTSD
LYZL4
SCAND2
EPHA5
VPS37A
1.95E-03
1.95E-03
1.95E-03
1.95E-03
1.95E-03
1.95E-03
1.95E-03
3.91E-03
7.39E-03
7.81E-03
7.81E-03
7.81E-03
7.81E-03
7.81E-03
7.81E-03
7.81E-03
1.56E-02
b,c,d
c
c
a,c
b
b,c,d
d
c
d
b,c
b,d
b
d
d
c
b,c,d
b,c,d
Shading indicates validated apigenin targets. ENSG, Ensembl gene; UPF3B, up-frame shift suppressor 3 homolog B.
*P value of log10(nICPGInput2) compared with log10(nICPGA-E2).
GO: a, GTPase activation; b, membrane; c, alternative splicing; d, others.
in breast cancer cell extracts (Fig. S4C). Our results show that
the inhibition of UGDH by avonols can be extended to
avones.
The third approach consisted of evaluating the interaction of
the identied targets expressed as GFP-tagged with apigenin in
human epithelial HeLa cells. Pull-down assays of lysates from
HeLa cells expressing hnRNPA2-GFP with either A- or C-beads
and immunoblotted with anti-GFP showed that hnRNPA2-GFP
specically binds the A- but not C-beads (Fig. 2A, line 2). Similar
results were obtained for ARHGEF1-GFP and GFPBCL2associated athanogene (BAG1), an antiapoptotic protein that
enhances the activity of B-cell lymphoma 2 (BCL2) (43) (Fig. S4
D, line 2 and E, line 2).
Collectively, our validation results underscore the value of PDSeq for the identication of apigenin targets. The nding that
most of the apigenin targets correspond to one of three main
categories (GTPase activation, membrane transport, and mRNA
metabolism/alternative splicing) has several interesting implica-
GRD
hnRNPA2
36 kD
forms. (C ) Different versions of recombinant
hnRNPA2GRD
afnity-puried GST-hnRNPA2 proteins were pulled
hnRNPA2C
down with A- or C-beads (indicated as A or C, reRRM
GRD
13 14
15 16
17 18 19 20
21 22 23 24
spectively). Pull-down assays (bound) and superBound Supernatant
Bound Supernatant
Bound Supernatant
natants fractions were resolved by SDS/PAGE and
analyzed by Western blot using anti-GST antibodies. Arrows indicate the correctly sized products; smaller bands present in some of the lanes correspond to
degradation products.
YGGG YGGG
Arango et al.
YGGG
APPLIED BIOLOGICAL
SCIENCES
Table 1. Cont.
0.1
0
0.2
0.1
0
0
1
5
10
25
50
100
800
400
0
470
530
590
Wavelength (nm)
0.5
0.4
1.9
0.3
0.2
1.8
1.7
0.1
1.6
0.1
1
10
GST-hnRNPA2 (M)
100
20
40
60
80
100
Apigenin (M)
1.6
1.5
1.2
Saturation
(l*[Apigenin]/A) x 10 -4
eI
Kp
Flavonoid (M)
YFP
hnRNPA2 C
1200
0
0.2
0.5
1
2
5
10
20
YFP/CFP
300
CFP
GST-hnRNPA2 (M)
300 370 440
Wavelength
0.3
260
pFLIP2-3-hnRNPA2 C His
Sp
GST
0.2
RFU
Absorbance
0.5
0.4
0.3
nI
0.6
Absorbance
0.8
1.0
0.5
0.4
K D = 2.66 +
- 1.09 M
0
K D = 22.99 +- 7.70 M
0.0
0
5
10
15
1/[hnRNPA2] x 105
20
E2158 | www.pnas.org/cgi/doi/10.1073/pnas.1303726110
50
Apigenin (M)
100
Arango et al.
R2
0.00012
0.00031
0.00002
0.01873
0.91532
0.873
0.880
0.916
0.815
0.897
0.73034
0.468
N.B.
0.10702
0.10737
0.96653
0.03077
0.99484
0.213
0.120
0.233
0.037
0.503
N.B.
N.B.
N.B.
N.B.
N.B.
KD (M)
22.99
131
126.6
27.13
60.88
7.70
78.89
60.18
12.24
24.14
Data represent mean SEM (n = 3). KD, dissociation constant; R2, coefcient of determination of saturation curves tted by nonlinear regression
(Materials and Methods).
*Statistical signicance of the YFP/CFP ratios over the tested avonoid concentration range (Fig. 3E and Fig. S7).
N.B., no binding.
and Fig. S7B). Consistent with -hnRNPA2 recognizing apigenin but not naringenin (Table 2 and Fig. S4A), neither naringenin
nor the related avanone eriodictyol affect the FRET of the
nanosensor (Table 2 and Fig. S7D). Suggesting that the binding
to hnRNPA2 is not limited to avones and consistent with the
fact that many biological activities of avones being shared by
avonols (5), quercetin and kaempferol also show a signicant
interaction with hnRNPA2 but with lower afnity than apigenin
(Table 2 and Fig. S7E). No binding of hnRNPA2 to avopiridol
or the isoavone genistein was detected, suggesting that these
biologically active compounds function through mechanisms distinct from apigenin (Fig. S7 C and F).
C
Conclusion
We describe here the comprehensive identication of the human
cellular targets of the avone apigenin, an abundant dietary
phytochemical with anticarcinogenic activities, by PD-Seq, a
high-throughput strategy that combines phage display with next
generation sequencing. The identied apigenin candidate targets
are not obviously enriched in ATP binding enzymes, which could
have been expected based on previous studies (44, 45); also, they
are not randomly distributed across multiple types of proteins.
Rather, most (121/160) of the candidate targets fall into one of
three categories: GTPase activation, membrane transport, and
mRNA metabolism/alternative splicing. This specicity of apigenin for specic types of proteins is revealing in terms of how
this avone may exert very specic cellular responses (60, 61).
Evidence of how phytochemicals perturb gene function is provided by the ability of apigenin to bind to the GRD of hnRNPA2
and inhibit its dimerization, which is essential for RNA binding
30
25
20
15
10
*
0
+
Boiled
GST-hnRNPA2
Boiled
6xHis-hnRNPA2
GST
30
25
20
15
*
10
5
0
DMSO
Api
Nar
PNAS PLUS
APPLIED BIOLOGICAL
SCIENCES
Apigenin
Luteolin
Quercetin
Kaempferol
Apigenin
6-C-glucoside
Apigenin
7-O-glucoside
Chrysoeriol
Naringenin
Eriodyctiol
Genistein
Flavopiridol
Flavonoid
MDA-MB-231
DMSO Api
Lut
MCF-10A
Nar
DMSO
c-FLIPL
6
c-FLIPS
1
100
c-FLIPS
80
Api
Isoform %
60
40
20
MDA-MB-231
DMSO Api
Lut
DMSO
MCF-10A
Nar
DMSO
Api
Isoform %
Caspase-9a
7
Caspase-9b
1
Api
50
Lut
Nar
Caspase-9b
40
30
20
10
DMSO Api
Lut
DMSO
MCF-10A
MDA-MB-231
Nar
DMSO
Api
Api
25
Lut
Nar
BIRC5-2b
2b
GAPDH
1
Isoform %
20
BIRC5-2b
BIRC5
15
10
5
0
DMSO
and hence, important for the participation of hnRNPA2 in alternative splicing. Indeed, we show that apigenin affects the
splicing of hnRNPA2 substrates in breast cancer cells. These
results may help explain how apigenin exerts an anticarcinogenic
activity by decreasing the inhibition of apoptosis, thereby increasing
the efcacy of chemotherapeutic drugs. This study offers a fresh
view on how dietary phytochemicals are likely to inuence the
systems network by impacting multiple (hundreds) cellular targets
with relatively low (micromolar range) afnity, moderately affecting
their activities (enzymatic or proteinprotein interaction). Thus, in
contrast to a pharmaceutical drug selected to have high afnity
and specicity, the effect of a dietary phytochemical would be
distributed across the entire network, resulting in a ne-tuning
effect, with a consequent long-term impact on health.
Materials and Methods
Preparation of Apigenin-Immobilized PEGA Beads. Apigenin was immobilized
to amino PEGA beads (EMD Biosciences). These beads are referred throughout
the text as A-beads. Acetylated PEGA beads were used as controls and are
referred throughout the text as C-beads. C-beads were loaded with acetic
anhydride (Ac2O) according to the procedure reported previously (62). Detailed information on the generation of beads can be found in SI Materials
and Methods.
Phage Display Screening. The phage display screening was performed using
a T7 Select Human Breast Tumor cDNA phage library (EMD Biosciences). The
original library was amplied by Plate Lysate Amplication according to the
manufacturers protocol. Preclearing of the amplied library was done
by incubating 2 mL T7 phage (109 pfu/mL) with 200 L (10 mg/mL) C-beads at
4 C overnight. The precleared phage suspension (1 mL) was incubated with
100 L 10 mg/mL A- or C-beads at 4 C overnight and washed 10 times with
1 mL buffer of 20 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween 20
followed by elution with 100 L 1% SDS for 10 min at room temperature.
Eluted fractions (5 L) were inoculated into 3 mL Escherichia coli Rosettagami B5615 (EMD Bioscience) host bacteria cells and incubated for 3 h at
37 C. Phage-infected cells were centrifuged at 800 g for 5 min, and
supernatants containing phage particles were used for the next biopanning
step. Phage titers for each biopanning step were evaluated by counting pfu
per milliliter according to the manufacturers protocol (EMD Biosciences).
Illumina GAII Second Generation Sequencing of cDNA Phage Libraries. Phage
DNA was isolated from the input and the elution fractions obtained in the
rst and second rounds of biopanning using either A- or C-beads (Fig. 1C)
(referred as AE or CE, respectively) by phenol/chloroform extraction and
amplied by PCR using T7 anking insert primers (Fig. 1D and Table S1). The
primers include three consecutive random nucleotides at the 5 regions to
help cluster recognition in Illumina GAII. Amplied PCR fragments were
used for preparation of Illumina libraries (Illumina) according to the manu-
E2160 | www.pnas.org/cgi/doi/10.1073/pnas.1303726110
Api
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Arango et al.
ALPHA. Dimerization of hnRNPA2 was determined by ALPHA using GSHacceptor and antiHis-donor beads according to the manufacturers instructions (Perkin-Elmer). Briey, 6xHis-hnRNPA2 (125 nM) was incubated with
125 nM GST-hnRNPA2 for 1 h at room temperature followed by the addition
of 20 g/mL GSH-acceptor and 20 g/mL antiHis-donor beads in 20 L TBS
buffer, pH 7.6, containing 1 mM DTT, 0.1 mM PMSF, and 2 g/mL each
chymostatin, pepstatin, antipain, and leupeptin and incubated for an additional 6 h at room temperature. Apigenin (100 M), naringenin (100 M), or
diluent control (DMSO) was added for 15 min at room temperature. Arbitrary uorescence units were determined using the EnSpire multimode plate
reader with ALPHA technology (Perkin-Elmer) and expressed divided by
1,000. Statistical signicance was determined by one-way ANOVA using
the GraphPad Prism software.
Cell Culture. Detailed information is in SI Materials and Methods.
Enzymatic Assays. To determine UGDH activity, MDA-MB-231 cells were
treated with 50 M apigenin, naringenin, or diluent DMSO for 3 h. Cells were
homogenized by douncing (10 strokes) in lysis buffer (10 mM TrisHCl, pH
8.7, 50 mM KCl, 1.5 mM MgCl2, 0.1 mM PMSF, 2 g/mL each chymostatin,
pepstatin, antipain, and leupeptin) and centrifuged at 20,000 g for 15 min
at 4 C. Cell lysates (250 g protein) were incubated in 200 L buffer containing 1 mM UDP-glucose, 100 mM sodium glycine, pH 8.7, and 1 mM NAD+
at room temperature. Activity was determined by assessing the change in
NAD+ absorbance at 340 nm for 30 min using the EnSpire multimode plate
spectrophotometer reader (Perkin-Elmer).
IDH3 activity was determined in mitochondria preparations from MDAMB-231 cells treated with 50 M apigenin, naringenin, or diluent DMSO as
control for 3 h. Mitochondria were isolated from 2 107 cells by dounce
homogenization (100 strokes) in 400 L mitochondria isolation (MI) buffer
containing 20 mM Tris, pH 7.2, 0.8 M sucrose, 40 mM KCl, 2 mM EGTA, 1 mg/
mL BSA, 0.1 mM PMSF, and 2 g/mL each chymostatin, pepstatin, antipain,
and leupeptin and centrifuged at 1,500 g for 10 min at 4 C. Pellets were
resuspended in 400 L MI buffer and centrifuged at 17,000 g for 30 min at
4 C. Pellets containing mitochondrial fraction were resuspended in 200 L
MI buffer and lysed by three rounds of freeze and thaw. Purity of the isolated mitochondria was veried by Western blot using antibodies against
cytochrome c, a specic mitochondrial marker, and GAPDH, a cytoplasmic
marker. IDH3 activity was evaluated by incubating 250 g mitochondrial
protein in 200 L buffer containing 100 mM K2HPO4, 100 mM KH2PO4, 8 mM
MgCl2, 500 M NAD+, and 2 mM sodium isocitrate, pH 7.6. Activity was determined by assessing the change in NAD+ absorbance at 340 nm for 30 min
using the EnSpire multimode plate reader.
Enzymatic units were calculated using the following formula: enzymatic
units = (A340 Vf d.f.)/(e mg l), where A340 is the change in absorbance
at 340 nm over time, Vf is the nal reaction volume, d.f. is the dilution factor, e
is the extinction coefcient of NAD+ determined to be 6.22, mg is the amount
of protein, and l is the light path estimated to be 0.68. Levels of statistical
signicance between treatments were determined by one-way ANOVA.
Arango et al.
PNAS PLUS
nonlinear regression using the GraphPad Prism. Levels of statistical signicance between means in FRET experiments were determined by oneway ANOVA.
APPLIED BIOLOGICAL
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Agricultural and Food Research Initiative Competitive Grant 2010-6511520408 (to E.G.) and National Institutes of Health (NIH)/National Heart, Lung,
and Blood Institute (NHLBI) Grant R01HL075040-01 (to A.I.D.).
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