Supplementary Information
FoxO3 coordinates metabolic pathways to maintain redox balance in neural stem cells
Hyeonju Yeo, Costas A. Lyssiotis, Yuqing Zhang, Haoqiang Ying, John M. Asara, Lewis C.
Cantley, and Ji-Hye Paik
Supplementary Figures and Legends
: 5 figures
Supplementary Materials and Methods
1
Yeo_Supplementary Figure S1
WT KO
A
WT KO
B
A
NES=2.071 p=0
WT KO
NES=1.609 p=0
C
NES=1.1404 p=0.025
D
Supplementary Figure S1.
The heat maps show the genes within enriched gene sets identified by the GSEA of differentially expressed genes in WT and FoxO3 KO NPCs. The KEGG pathways, (A) ArginineProline Metabolism, (B) Glycolysis and Gluconeogenesis and (C) Pentose Phosphate pathway, were represented to be downregulated in FoxO3 KO NPCs. (D) Ingenuity pathway
analysis of differentially expressed genes in WT and FoxO3 KO NPCs.
2
Yeo_ Supplementary Figure S2
B
Basal
respiration
**
450
300
150
0
WT1
WT2
WT3
KO1
KO2
8
*
6
4
2
0
KO3
Maximum
respiration
*
10
ΔpO2 /hr /5X105cells
Relative mitochondria DNA
copy number (Cox2/β-globin)
A
WT FoxO KO
WT FoxO KO
+ FCCP
C
Cell Count
400
No stain
WT
FoxO KO
300
200
100
0
100
101
102
103
104
Mitosox (PE-Texas Red-A)
*
0.02
0.01
0
WT
FoxO KO
0.075
0.05
*
0.025
0
FoxO KO
WT
Relative MnSod expression
0.03
Relative Catalase1 expression
Relative Sesn3 expression
D
0.06
0.04
0.02
0
WT
FoxO KO
Supplementary Figure S2.
(A) The mitochondrial DNA copy numbers in WT and FoxO3 KO NPCs were measured by
qPCR analysis and data was shown as mean ± SE. (B) The oxygen consumption, Basal and
maximum respiration, was analyzed in WT and FoxO3 KO NPCs without or with treatment
of 1 μM FCCP, respectively. Data was shown as mean ± SD. (C) The mitochondrial superoxide was measured by Mitosox staining and flow cytometry. (D) The mRNA expressions of
Sesn3, Catalase1 and MnSod in WT and FoxO3 KO NPCs were measured by RT-qPCR analysis and data was shown as mean ± SE.
3
Yeo_ Supplementary Figure S3
0
**
WT
FoxO KO
0.003
n.s.
0.002
0.001
0
WT
FoxO KO
Relative GS expression
(fold change)
0.002
0.00012
n.s.
0.00008
0.00004
0
FoxO KO
WT
0.15
Relative Glut1 expression
0.004
Relative GLS2 expression
0.006
Relative LAT1 expression
Relative ASCT2 expression
Relative FoxO3 expression
A
n.s.
0.1
0.05
0
FoxO KO
WT
1.2
0.9
*
0.6
0.3
0
WT
FoxO KO
0.45
n.s.
0.3
0.15
0
WT
FoxO KO
B
Cell Count
FL1-DCF-DA
90
60
30
0
200
**
120
WT
FoxO3 KD
293T
No stain
WT 293T
FoxO3 KD 293T
100
0
100 101
102
103
104
DCF (FITC-A)
Supplementary Figure S3.
(A) The mRNA expression of FoxO3, Gls2, GS, Asct2, Lat1 and Glut1 in WT and FoxO3
KO NPCs were measured by RT-qPCR analysis and data was shown as mean ± SE. (B) Intracellular ROS was measured by DCF-DA staining in 293T and 293T FoxO3 KD cells.
**p<0.005
4
Yeo_ Supplementary Figure S4
B
32wk Age
7wk
NAC P.O.
GSH/GSSG
0.032
C
KO
WT
*
*
nitrotyrosine
A
0.024
0.016
CONTROL
NAC
0
CON
Fold change
Nitrotyrosine + spot intensity
0.008
NAC
80
WT
**
0.3
0
NAC
Water
KO
WT
**
40
0
KO
CONTROL
0.6
DCX+ neurogenesis
DCX
Sox2+ NSC in SVZ
SOX-2
120
WT
0.9
F
E
D
1.2
160
NAC
KO
120
80
40
0
CON
WT
**
CON
NAC
KO
NAC
Supplementary Figure S4.
(A) NAC administration scheme. After feeding NAC to WT and FoxO1/3/4 KO mice (B)
GSH/GSSG was assayed in the brain tissues. Immunohistochemistry was performed for nitrotyrosine in CA2 region of the brain (C) and Sox2 and DCX in the SVZ of the brain (D). Microscopic fields of comparable regions were examined at a magnification of 200X. Sox2 (E),
DCX (F) positive cells were counted in the same area of the brain and nitro-tyrosine spot intensity was counted shown as a graph. Error bars represent ± SD values of the mean. Error
bars represent ± SE values of the mean. *p<0.05, **p<0.01
5
Yeo_ Supplementary Figure S5
GLS Activity mU/mg/hr
A
*
*
50
40
GLS1
20
FoxO3
10
0
HA
DN-AKT
GFP
B
DN-AKT
GFP
FoxO KO
GAPDH
*
*
50
*
WT
40
FoxO KO
GFP Ca- GFP CaFoxO3
30
FoxO3
GLS1
20
FoxO3
10
0
FoxO KO
DNDNGFP
GFP
AKT
AKT
30
WT
GLS Activity mU/mg/hr
WT
*
GAPDH
GFP
Ca-FoxO3
WT
GFP
Ca-FoxO3
FoxO KO
Supplementary Figure S5.
GLS activity was assayed in either (A) Ad-GFP or DN-AKT expressing NPCs and (B) caFoxO3 expressing adenovirus infected FoxO3 KO NPCs. The data was shown as mean ± SD
of fold change. The expression of DN-AKT and ca-FoxO3 were confirmed by immunoblots.
*p<0.05
6
Supplementary Materials and Methods
Intracellular ROS measurement
Intracellular ROS was detected using an intracellular ROS dye, dichlorodihydrofluoresein
(DCF-DA). NPCs were incubated with 10 μM DCF-DA for 30 min at 37°C. The level of fluorescent adduct was determined by flow cytometry with excitation at 488nm. To assess the
effect of Gln metabolites on intracellular ROS accumulation, NPCs were cultured in 25 or 1
mM glucose and 2 or 0.2 mM Gln. To determine the effect of Gln metabolites on ROS, Glnstarved NPCs were treated with 4 mM Glu or 2 mM cell permeable GSH for 16 h prior to the
analysis. The same conditions were used to measure intracellular NADP/NADPH using an
assay kit (Abcam). For intramitochondrial superoxide determination, MitoSox (Invitrogen)
dye was added to NPCs and dye intensity was measured by flow cytometry.
Immunoblot analysis
Cells were lysed with RIPA buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1 % Nonidet P-40,
0.5 % sodium deoxycholate, 1 mM EDTA, 0.1 % SDS) containing complete protease and
phosphatase inhibitors. Protein concentration was determined by Bradford assay and 20 μg of
proteins was used for SDS-PAGE. Immunoblots were incubated with the following antibodies: FoxO3, HK1, HK2, pAKT, AKT, PKM2, total PKM, PIK3CA, pAMPK, p-mTOR,
mTOR, Rictor, Raptor, p-S6, p-p70S6K, p-eIF4E, pULK, GLUD1, GAPDH, HA (Cell Signaling), PKLR (Novous), V5, Flag (Sigma), GLS1 (ProteinTech), and alpha-tubulin (DHSB).
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Under reducing condition of SDS-PAGE, cell lysates were mixed with 6x SDS-Sample buffer
(375 mM Tris-Hcl pH6.8, 6 % SDS, 48 % glycerol, 9 % -mercaptoethanol, 0.03 % Bromophenol) and heat denatured for 10 min.
qRT-PCR analysis
RNA was harvested using GeneJET RNA purification kit (Fermentas) and treated with RQ1
RNase-free DNase (Promega). Then, one microgram of total RNA was reverse transcribed
with Maxima® first strand cDNA synthesis kit (Fermentas). Quantitative RT-PCR was performed on cDNA samples using the Power SYBR® Green Master mix and was performed
the qPCR on the StepOnePlusTM Real Time PCR System (Applied Biosystems). The relative
mRNA level was presented as values of 2^[Ct(β-actin) – Ct(gene of interest)]. The sequences
of primers are listed as below and PPP pathway related genes are described previously (Ying
et al., 2012).
Gene ID
Sequence
FoxO3
F:CTGGGGGAACCTGTCCTATG
R:TCATTCTGAACGCGCATGAAG
GLS1
F: CTACAGGATTGCGACATCTGAT
R: ACACCATCTGACGTTGTCTGA
GLS2
F: CGTCCGGTACTACCTCGGT
R: TGTCCCTCTGCAATAGTGTAGAA
PKM1
F: GCTGTTTGAAGAGCTTGTGC
R: TTATAAGAGGCCTCCACGCT
PKM2
F: TGTCTGGAGAAACAGCCAAG
R: TCCTCGAATAGCTGCAAGTG
ASCT2
F: CATCAACGACTCTGTTGTGACC
R: CGCTGGATACAGGATTGCGG
LAT1
F: CTACGCCTACATGCTGGAGG
R: GAGGGCCGAATGATGAGCAG
GLUD1
F: CCCAACTTCTTCGATGGTGG
R: AGAGGCTCAACACATGGTTGC
SLC7A1
F: CAGTTCGGCTATAACACTGGTG
R: GCCCCCGACAGAGAAGATG
ACSS1
F: GTTTGGGACACTCCTTACCATAC
R: AGGCAGTTGACAGACACATTC
8
ACSS2
F: AAACACGCTCAGGGAAAATCA
R: ACCGTAGATGTATCCCCCAGG
PIK3CA
F: CCACGACCATCTTCGGGTG
R:ACGGAGGCATTCTAAAGTCACTA
Rictor
F: ACAGTTGGAAAAGTGGCACAA
R: GCGACGAACGTAGTTATCACCA
Glut1
F: CAGTTCGGCTATAACACTGGTG
R: GCCCCCGACAGAGAAGATG
Chromatin immunoprecipitation
5 millions of NPCs were cross-linked with 1 % formaldehyde followed by quenching with
125 mM glycine. The cells were lysed in lysis buffer containing 1 % SDS, 10 mM EDTA,
and 50 mM Tris (pH 8.1), then sonicated 15 times for 30s. The sheared DNA was diluted in
dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1),
and 167 mM NaCl), pre-cleared and incubated with anti-rabbit IgG and anti-FoxO3 IgG
overnight at 4 °C and collected with protein G gamma plus sepharose beads. Precipitates
were washed sequentially using EZ ChIP wash buffers and DNA was purified using a PCR
purification kit (Qiagen). TRANSFAC algorithm was used to predict the FoxO3 binding site
on regulatory region of target genes (up to 5000 bp upstream of TSS) with minimal false positive stringency. Two sets of primers per each target were designed to examine FoxO3 binding. Enrichment was calculated based on Ct values of qPCR and plotted as fraction of total
input. For negative control, we used chromosome 6 genomic scaffold region and the sequences of primers are as below.
Gene ID
Negative
Ddit4
GLS1-1
Sequence
F: GGGGGATAATGATTGCAAAA
R: GCGTGGACAGAGATGTAGGC
F: CTTTCAGCAGCTGCCAAGGTC
R: CAGAAGCTAGGGGTACCTTTCTC
F: GGATCTGCAGTATTTTGCTTTCA
R: GCCAGAGTAAGGGTGGGATCT
9
GLS1-2
F: TCGTTGTTCCTACGCTCAAA
R: CCCTCTCCCAAATGTGCTTA
Glud1-1
F: GCAGGCCGGAGAATCTTTT
R: AATTACATCAATATAAACAGTGGTTGA
Glud1-2
F: CGCTGAAGTTGCCTACAACA
R: ACTCATCCCGAATCCGACT
Pgd-1
F: GCACGAGGTACACGGACATA
R: CGCACAGAAAGAGAAAACGA
Pgd-2
F: CGCTTGGCTCTTTACTTGCT
R: GGCAGGAGTCAAGTCTGTCC
RPE-1
F: CGGAATGCTGCAATTAATGA
R: CTCCTCCTGACCCCTTCTTC
RPE-2
F:AAGAAAGGGTAATGGGGTTGA
R: TTCTACGGGTTGATCAGCTAGAC
TALDO-1
F: TCAACCTCGCCTAGTTGCTT
R: TTCTTTGAATATGCTGGCTCAA
TALDO-2
F: GGGGTCTGAGGCTTACTGAG
R: TCAGGTCGGAGTTCTCTTC
PDK4-1
F: GGAGGTCAATATTGGGCAGT
R: TTTGTCACTAGCAGGCACCA
PDK4-2
F: GCGGTGAGATTCTTGGAAAC
R: CCAGGTCGCTAGGACTTCAG
ACSS1-1
F: TCAGCAGCTGGTAACCAAAA
R: CCTGTACAACCATGCCACAC
ACSS1-2
F: CCGTGTACTTGCAGCTTCTG
R: GTTTTGTTGTGCGCCCAGT
ACSS2-1
F: TCTGAAGGATCCTCCTCGTC
R: GGCACAGATCCCAATATGA
ACSS2-2
F: CCGTAACCCAACCCTTGTC
R: GTGGAGTGATGGGGTGTAGC
Pyruvate kinase activity assay
Pyruvate kinase activity was determined by a pyruvate oxidase-based assay kit (Biovision),
according to the manufacturer’s instruction. Briefly, NPCs were extracted with 4 volumes of
the PK assay buffer and cleared by centrifugation. For oxidant treatment, cell lysates were
treated with 250 μM of diamide (Sigma) for 15 min at 4°C. Reaction was performed in a dark
walled 96 well plate, with 50 μg lysates in 50 μl PK assay buffer containing mixture of substrates, pyruvate oxidase, and probe. Pyruvate kinase activity was determined by changes in
OD at 570 nm. Basically, PEP and ADP were catalyzed by PK to generate pyruvate and ATP,
10
and the generated pyruvate is oxidized by pyruvate oxidase to produce color.
Glutamate Dehydrogenase activity assay
Glutamate dehydrogenase (GLUD) activity was determined by a glutamate dehydrogenase kit
(Biovision), according to the manufacturer’s instruction. Briefly, NPCs were lysed with
GLUD assay buffer and cleared by centrifugation. Reaction was performed in a dark walled
96 well plate, with 40 μg lysates in 25 μl GLUD assay buffer containing mixture of glutamate
and developer. As GLUD converted glutamate to KG, GLUD in lysates consumed glutamate as substrate and the generated NADH was quantified colorimetrically. GLUD activity
was determined by changes in OD at 450 nm.
Hexokinase activity assay
Hexokinase enzyme activity was assessed by the coupling of glucose-6-phosphate production
with NADH production in the presence of glucose-6-phosphate dehydrogenase (G6PDH).
NPCs were extracted using PK assay buffer and used 50 μg of lysates for assay. After samples were mixed with assay buffer containing 25 mM triethylamino-Cl, 15 mM MgCl2, 5 mM
ATP, 1 mM NAD, 1 mM DTT, 0.5 mM glucose, 0.45 mM KCN and 1.2 units/ml G6PDH
(1KU, Glucose-6-phosphate Dehydrogenase, from L. mesenteroides, Sigma), the rate of
NADH formation was determined as OD at 340 nm.
Glutaminase activity assay
Glutaminase activity was determined according to the method of Pinkus and Windmueller.
11
Briefly, NPCs were resuspended in glutamate assay buffer (pH 8.1) and Gln was added to the
supernatant at 17 mM. The mixture was incubated for 1 h at 37 ◦C and the reaction was
stopped by adding 0.2 volume of 10 % trichloroacetic acid. The amount of glutamate produced was determined by glutamate assay kit (BioVision). The GLS inhibitor 968 is a kind
gift from Dr. Richard Cerione. BPTES Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl
sulfide (Sigma) was dissolved in DMSO.
NADP/NADPH assay
The ratio of NADP and NADPH was determined by a NADP/NADPH assay kit (Abcam),
according to the manufacturer’s instruction. Briefly, NPCs were cultured both high (25 mM)
and low (5 mM) glucose and with or without 2mM Gln and lysed with extraction buffer by
two freeze/thaw cycles. Reaction was performed in a dark walled 96 well plate with 20 μg
lysates in 50 μl assay buffer containing NADP Cycling buffer and enzyme mixture. After incubation for 5 min to convert NADP to NADPH, then NADPH developer was added to
measure the conversion of NADP to NADPH. The ratio was determined by changes in OD at
450 nm.
Glutathione assay
The ratio of GSH and GSSG was determined by glutathione assay kit (Biovision), according
to the manufacturer’s instruction. Briefly, NPCs and brain tissue were lysed and mixed with
pre-chilled perchloric acid followed by KOH neutralization and the reaction with OPA probe
12
was performed in a dark walled 96 well plate. The reaction was determined by changes in
fluorescence Ex/Em = 340/420 nm.
Metabolite analysis by targeted liquid-chromatography-tandem mass spectrometry
24 h prior to each experiment, 1x106 cells were plated in 6 cm dishes in regular growth media.
Importantly, we used WT and FoxO3 KO NPCs with comparable growth kinetics and retaining >99% Nestin expression to avoid an indirect consequence of compromised proliferation
or aberrant differentiation which can influence anaplerosis of Gln. Fresh media were replaced
2 h before the experiment. For metabolite collection, media from biological duplicates was
fully aspirated from cells and 4 ml of 80 % (v/v) methanol was added. Cells and the metabolite-containing supernatants were collected into conical tubes. Insoluble material in lysates
was centrifuged at 2,000xg for 15 min, and the resulting supernatant was SpeedVac-dried.
Samples were re-suspended using 20 μL HPLC grade water and 5 μL injections for targeted
tandem mass spectrometry (LC-MS/MS) as previously described (Ying et al., 2012; Yuan et
al., 2012) Briefly, positive/negative ion polarity switching with selected reaction monitoring
(SRM) was used to target more than 250 polar metabolites. A 5500 hybrid QTRAP mass
spectrometer (AB/SCIEX) was coupled to a Prominence HPLC (Shimadzu) using a 4.6 mm x
10 cm Amide XBridge HILIC chromatography column at pH=9.0 (Waters) at 400 μL/min.
Isotope labeling for glutamine incorporation assay
Glutamine-free DMEM/F12 was supplemented with uniformly labeled 2 mM [13C5]Gln
13
(Cambridge Isotope Laboratories). Cells were maintained in the labeled Gln containing media
for 24 h and switched into freshly prepared same media for 2 h prior to harvesting. Metabolite fractions were collected and analyzed by targeted LC-MS/MS, as described above.
Pentose phosphate pathway (PPP)-dependent glucose oxidation to CO2
PPP activity was measured using an adaptation of previously published procedures (Ying et
al., 2012). Briefly, cells were plated in 96-well plates 24 h prior to the experiment. Media
were supplemented with 5 μCi/ml of [1-14C]-glucose (NEC043X001MC, specific activity 4560 mCi/mmol) in order to measure the release of 14CO2 from [1-14C]-glucose which provides
a quantitative measure of flux through the PPP enzyme, 6-phosphogluconate dehydrogenase.
The wells were overlaid with Ba(OH)2 impregnated 3MM Whatman paper and plates were
incubated at 37 ◦C 5% CO2 to capture released 14CO2. The Whatman paper was removed, then,
placed in an acetone bath, air-dried and incubated at 110 ◦C for 5 min and radioactivity was
measured in a Perkin Elmer Tri-Carb liquid scintillation counter. PPP-dependent CO2 production was calculated as the difference between 14CO2 derived from [1-14C]-glucose and 14CO2
derived from [6-14C]-glucose (NEC045X050UC specific activity 50-62 mCi/mmol) which is
a TCA cycle-dependent CO2 production from glucose.
NAC feeding
FoxO WT (FoxO1/3/4L/L) and null (hGFAP-Cre+: FoxO1/3/4L/L) mice (Paik et al, 2009) were
put on drinking water containing 40 mM NAC (Sigma) from 7 to 32 weeks of age. Water was
14
replaced weekly (Reliene et al, 2006).
Immunohistochemistry
Formalin-fixed, paraffin-embedded 5 micron sections were used. For stains of brain, sections
were processed in standard method and incubated with primary antibodies to Goat-anti-Sox2
or goat-anti-doublecortin (DCX, C-18) from Santa Cruz Biotechnology, and anti-Nitrotyrosine from Alpha Diagnostics, and further processed by Vector Elite ABC peroxidase kit
followed by developing with DAB substrate and counterstained with hematoxylin. Quantitation of DAB positive staining was performed by ImageJ software. Briefly, 20x images were
imported and red channel was selected by splitting channel option. Images were inverted and
converted to binary prior to measuring integrated densities of the region of interest.
Oxygen consumption measurement
To measure oxygen consumption rate, SDR SensorDish® Reader instrument (PreSens) was
utilized. Cells were plated in OxoDish® containing an oxygen sensor at the bottom of each
well. For maximum respiration capacity, cells were treated with 1 μM FCCP .
Measurement of mitochondria DNA copy number
Mitochondrial DNA copy number was quantified by determining the ratio of mitochondrial
cytochrome C oxidase (Cox) 2 (F: GCCGACTAAATCAAGCAACA, R: CAATGGGCATAAAGCTATGG) to nuclear intron of β-globin (F: GAAGCGATTCTAGGGAGCAG, R:
GGAGCAGCGATTCTGAGTAGA) by quantitative real-time PCR.
Colocalization study
15
Localizations of HK2 to mitochondria and mTOR to lysosomes were determined by costaining of Mitotracker and HK2 or LAMP1 and mTOR. The level of colocalization was determined by analysis of overlaid images with WCIF Image J colocalization module (colocalization finder) with a fixed threshold after background subtraction. Percent of both green and
red positive spots from total green positive signals was calculated.
16