Supplementary Revised01032020 FINAL copyNwosu et al. B. RT-qPCR validation of selected genes in...
Transcript of Supplementary Revised01032020 FINAL copyNwosu et al. B. RT-qPCR validation of selected genes in...
Nwosu et al.
SUPPLEMENTARY DATA
Severe metabolic alterations in liver cancer lead to ERK pathway activation and drug resistance
Figure S1. Glutamine deprivation impedes proliferation
A. MTT assay showing the proliferation of 10 HCC cell lines after 48h culture in media without glutamine (- Gln) (in quadruplicates).
B. Trypan blue viability assay of HUH7 and HLE cells after 72h of Gln deprivation, n=3 per group.
C. Continuous oxygen consumption assay for 5 days at 10 minutes intervals, n=3. Complete medium was introduced in each well after 72h (shown on the graph as Day 4). % a.s – percentage of air saturation.
D. Phase contrast image of freshly isolated mice hepatocytes cultured for 48h with or without Gln. On the right, CellTitre Glo viability assay. Error bars indicate mean±SD.
C.
A.
D.
HU
H7
HEP
G2
HEP
3BPL
CH
UH
1H
LE HLF
SNU
449
SNU
398
SNU
4750
50
100
150
Rel
ative
pro
lifera
tion
CM- Gln
Well diff. Poorly diff.
Ce
ll n
um
be
r (1
03
)
H U H 7 H L E 0
5 0
1 0 0
1 5 0 C M- G ln
**** Via
bili
ty (
%)
H U H 7 H L E 0
5 0
1 0 0
1 5 0 C M- G lnB.
M o u se H e p a to cy te
Via
bili
ty A
ssa
y(%
CM
)
C M - G ln
6 0
8 0
1 0 0
1 2 0
Nwosu et al.
Figure S2. Glutamine deprivation severely alters metabolism
A. Mass isotopologue distribution showing glucose carbon contribution to glutaminolysis intermediates, n=3 per group.
B. RT-qPCR showing the effect of Gln deprivation of glucose transporter 1 SLC2A1 and the mitochondrial pyruvate carrier 1 MPC1 in HUH7 and HLE cell lines after 48h (n = 3). Where indicated, statistical significance are: * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
C. Gene set enrichment plot showing pathway alterations derived from microarray of Gln-deprived HLE cells.
A. B.
C.
G lu ta m a te
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
M4
M5
0
5 0
1 0 0
1 5 0C M- G ln
****
****
****HU
H7
G lu ta m a te
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
M4
M5
0
5 0
1 0 0
1 5 0 C M- G ln
********
****
****
****
A sp a rta te
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
M4
0
5 0
1 0 0
1 5 0 C M- G ln
**
***
A sp a rta te
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
M4
0
5 0
1 0 0
1 5 0 C M- G ln
*
M ala te
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
M4
0
5 0
1 0 0
1 5 0 C M- G ln
****
****
M ala te
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
M4
0
5 0
1 0 0
1 5 0 C M- G ln
***
****
HLE
S L C 2 A 1 (G L U T 1 )
Re
l.m
RN
A le
vel
H U H 7 H L E 0
1
2
3 C M- G ln
* * * *
* *
M PC 1
Re
l.mRNA
leve
l
H U H 7 H L E 0
1
2
3
4
5 C M- G ln
* *
* * *
a-Ketoglutarate
13C
-glu
cose
labe
ling
(%)
M0 M1 M2 M3 M4 M50
50
100
150 CM- Gln
****
****
****
a-Ketoglutarate
13C
-glu
cose
labe
ling
(%)
M0 M1 M2 M3 M4 M50
50
100
150 CM- Gln
****
****
****
****
Nwosu et al.
Figure S3. Metabolic perturbation induces ERK pathway and oxidative stress genes
A. Heatmap of an independent microarray dataset showing the expression of the ERK pathway genes differentially expressed in the Gln-deprived HLE dataset. The heatmap includes AREG, which did not emerge as significantly changed in HLE dataset, but initially identified in HEPG2 cell dataset (not included) and is upregulated in RT-qPCR. HLE dataset accession number – GSE123062; MDAMB231 is a Gln-dependent breast cancer cell line.
H U H 7 H L E 0
1
2
3
4
N FE 2L2
Re
lativ
emRNA
leve
l C M- G ln
H U H 7 H L E0
1
2
3
4
H M O X 1
Re
lativ
emRNA
leve
l
C M- G
lnC M
- Gln
C M- G
ln0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
H L E
s ic o ns iN R F 2
N R F 2 H M O X 1 P S A T 1
H U H 7 H L E0 .0
0 .5
1 .0
1 .5
2 .0
N Q O 1
Re
lativ
emRNA
leve
l
C M- G
lnC M
- Gln
C M- G
ln0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
H U H 7
Re
lativ
emRNA
leve
l s ic o ns iN R F 2
N R F 2 H M O X 1 P S A T 1
H U H 7 H L E0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
SO D 2
Re
lativ
emRNA
leve
l
H U H 7 H L E0
2
4
6
P S A T 1
Re
lativ
emRNA
leve
l
A.
C.
0
5
1 0
1 5
M XD 1
Re
lativ
emRNA
leve
l
* * * *
0
1
2
3
G LS
HU
H7
Re
lativ
emRNA
leve
l
* * *
0
2
4
6
G LS
HL
E
Re
lativ
emRNA
leve
l* * * *
* * * *
* *
0
1
2
F A S N
Re
lativ
emRNA
leve
l
* * * *
0
1
2
F A S N
Re
lativ
emRNA
leve
l
* * *
0
1 0
2 0
3 0
4 0
A R EG
Re
lativ
emRNA
leve
l
* * * *
0
5
1 0
1 5
2 0
2 5
A R EG
Re
lativ
emRNA
leve
l
* *
* * *
* * * *
0
5
1 0
1 5
M YC
Re
lativ
emRNA
leve
l
*
* * * *
0
2
4
6
M YC
Re
lativ
emRNA
leve
l
* * * *
0
1
2
M XD 1
Re
lativ
emRNA
leve
l
U T- G lnA S P NA O A
Metabolic genes ERK pathway genes
B.
Nwosu et al.
B. RT-qPCR validation of selected genes in metabolism (FASN, GLS) and ERK pathway (MYC, AREG, MXD1) that are differentially expressed in Gln-deprived HLE cells. UT – untreated; ASPN – 1U asparaginase; AOA – 2.5 mM aminooxyacetate (a pan-transaminase inhibitor). Cells were cultured for 24h. Statistical significance * P<0.05, **P<0.01, ***P<0.001, ****P<0.000 relative to untreated.
C. RT-qPCR of NRF2-oxidative stress genes and serine pathway gene PSAT1 along with the effect of NRF2 knockdown after 24h. NRF2 – alias for NFE2L2.
Nwosu et al.
Figure S4. High serine load selectively affects Gln-dependent cells
A. Phase contrast microscopy of HCC cell lines cultured with high serine, methionine and glycine – 72h.
B. Image showing Gln-deprived HLE cell detachment 48h after serine supplementation. Representative of >3 experiments.
C. Image showing no effect of high serine supplementation on primary mouse hepatocytes. D. MTT proliferation assay showing serine supplementation effect on HUH7 and HLE cells
when cultured in complete media, 48h. * indicates concentrations that had significant impact on HLE cells relative to media with no additional serine supplementation.
E. Clonogenic assay following 7 days culture in complete or serine-free media.
B.
C.-Gln + SER [20mM]
Mouse Hepatocyte
-Gln
HLE
CM
HUH7
- Gln
CM+10mM SER
- Gln+10mM SER
CM+20mM SER
- Gln+20mM SER
A.
0 1 2 4 10 200
50
100
150
CM
Serine [mM]
Rela
tive
prol
ifera
tion HUH7 HLE
*
D.
E.
Nwosu et al.
H L E
S e rin e
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
0
5 0
1 0 0
1 5 0 C M- G ln
* * * *
* * * *
H U H 7
S e rin e
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
0
5 0
1 0 0
1 5 0C M- G ln
* * *
* * ** * *
* * *
CM
CM+1mM Ser
- Gln
- Gln+
1mM Ser
0.0
0.5
1.0
1.5
Aspartate
Rel
ative
leve
l (in
trace
llula
r)
CM
CM+1mM Ser
- Gln
- Gln+
1mM Ser
0.0
0.5
1.0
1.5
Malate
Rel
ative
leve
l (in
trace
llula
r)
*
CM
CM+1mM Ser
- Gln
- Gln+
1mM Ser
0.0
0.5
1.0
1.5
Glutamate
Rel
ative
leve
l (in
trace
llula
r)
*
CM- G
ln- S
er
- Gln/
Ser0
50
100
150
Malate
Rel
ative
leve
l (in
trace
llula
r)
****
CM- G
ln- S
er
- Gln/
Ser0
50
100
150
Aspartate
Rel
ative
leve
l (in
trace
llula
r)
**
CM- G
ln- S
er
- Gln/
Ser0
50
100
150
Glutamate
Rel
ative
leve
l (in
trace
llula
r)
CM- G
ln- S
er
- Gln/
Ser0
50
100
150
Malate
Rela
tive
leve
l (ex
trace
llula
r)
**
CM- G
ln- S
er
- Gln/
Ser0
50
100
150
AspartateRe
lativ
e le
vel (
extra
cellu
lar)
*
CM- G
ln- S
er
- Gln/
Ser0
50
100
150
Glutamate
Rela
tive
leve
l (ex
trace
llula
r)
HUH7 HLE 0
5
10
15
pERK1
pico
gram
/µg
prot
ein CM - Gln
- Gln+SER (5 mM)
HUH7 HLE 0
10
20
30
40
pERK2
pico
gram
/µg
prot
ein CM - Gln
- Gln+SER [5 mM]
A.
C.
B.
E.
D.
F.HU
H7HL
E
UT
SER
[20
mM
]
U012
6 [2
0 µM
]
SER
+U0
126
SER
+ER
LO [2
0 µM
]
SER
+SO
RA
[2.5
µM
]
-Gln
H.
HLE HLFHUH7 PLC SNU398
pERK
ERK
!-Tubulin
44/42
44
52
Gln + - + - - + + - + - - ++ - + - - + + - + - - + + - + - - +
Serine + + - - * * + + - - * *+ + - - * * + + - - * * + + - - * *
~kDa
G.
Nwosu et al.
Figure S5. High intracellular serine contributes to the ERK pathway induction A. Mass isotopologue distribution of 13C-glucose carbon in serine. Error bars indicate mean
± SD, n=3 samples. B. Intracellular metabolite quantification of the indicated metabolites in HLE cells
supplemented with serine in complete media or upon Gln deprivation (24h, n=3 per group).
C. Extracellular metabolite quantification in HLE cells deprived of Gln, serine or both (24h, n=3 per group).
D. Intracellular metabolite quantification in HLE cells deprived of Gln, serine or both (24h, n=3 per group).
E. Heatmap showing metabolites altered in Gln-deprived HLE cells in the experiment with serine deprivation. Figure S5C – D are parallel experiments, i.e. culture media and the corresponding cell lysates were collected from each plate well for analysis.
F. Phosphoprotein array showing pERK expression after 48h. Error bars indicate mean±S.E.M, n=5 per group, from two biological replicate experiments.
G. Western blot of HCC cell lines cultured with high serine or with no serine in complete media and in Gln-free media, after 24h. 5mM serine where supplemented.
H. Crystal violet staining of HUH7 and HLE cells supplemented with serine in Gln-deprived media and treated with pERK/MEK inhibitor (U0126), epidermal growth factor receptor inhibitor (Erlotinib, abbreviated ERLO) and multi-kinase inhibitor Sorafenib (abbreviated SORA). Cells were treated for 48h, followed by rescue with complete media for 3 days (HUH7) or 5 days (HLE).
Nwosu et al.
Figure S6. Inhibition of ERK pathway reverses Gln deprivation-induced signaling and inflammatory components
A. GSEA plot from HLE microarrays, showing molecular hallmarks that were induced by Gln deprivation but suppressed in the Gln-deprived HLE cells when treated with U0126, 20µM.
B. Heatmap showing the effect of U0126 on the ERK pathway-related genes that were differentially induced or suppressed upon Gln deprivation in HLE cells.
A. -Gln+U0126-Gln
B.
Nwosu et al.
Figure S7. Inhibition of ERK pathway enables proliferation in impaired metabolic state
A. MTT assay proliferation assay following treatment of HUH7 and HLE cells with MYC inhibitor 10058-F4 in complete media (left) and Gln-free media (right). The HLE cell data is representative of 2 repeated experiments.
H U H 7R
ela
tive
pro
life
ratio
n(%
of
UT
)
02
55
01
000
5 0
1 0 0
1 5 0
1 0 0 5 8 -F 4 (µ M )
*
****
F.
C.
Re
lativ
e p
rolif
era
tion
C M - G ln0
1
2 s iC O Ns iM X D 1
* * * * * * *
B.
H U H 7
Re
lativ
e p
rolif
era
tion
C M - G ln0
5 0
1 0 0
1 5 0
H L E
Re
lativ
e p
rolif
era
tion
C M - G ln0
5 0
1 0 0
1 5 0U TE R L O + U 0 1 2 6E R L O + S O R AU 0 1 2 6 + S O R A
** *
*
pAKTHUH7
LY294002 [µM]Gln
--
-+
20+
20-
50+
50-
-+
--
20+
20-
1h 24h
AKT
pAKTHLE
AKT
A. H L E
Re
lativ
e p
rolif
era
tion
(% o
f U
T)
02
55
01
000
5 0
1 0 0
1 5 0
1 0 0 5 8 -F 4 [µ M ]****
****
MXD1
Rel
.mRNA
leve
l
NT
siMXD1
sictrl
0.0
0.5
1.0
1.5
****
HLE
Rel
ative
pro
lifera
tion
(% o
f UT)
0
50
100
150
--
-50
-100
* *
HUH7
Rel
ative
pro
lifera
tion
(% o
f UT)
0
50
100
150
*
****
Gln10058-F4 [µM]
--
-50
-100
HLE (CM)
Rel
ative
pro
lifera
tion
(% o
f UT)
0
50
100
150
--
Erlotinib [20µM]U0126 [µM]
+-
-10
-20
+10
+20
HLF (CM)
Rel
ative
pro
lifera
tion
(% o
f UT)
0
50
100
150
--
Erlotinib [20µM]U0126 [µM]
+-
-10
-20
+10
+20
HLE (- Gln)
0
50
100
150
200
--
+-
-10
-20
+10
+20
**D18.2%
HLF (- Gln)
0
50
100
150
200
--
+-
-10
-20
+10
+20
********D40.6%D.
E.
HEP
3BPL
CH
UH
1SN
U44
9SN
U39
8SN
U47
50
50
100
150
200
250
Combination: EGFR/pERK inhibition: CM
Rel
ative
pro
lifera
tion UT
ERLO [20µM]U0126 [10µM]ERLO+U0126
Poorly diff.Well diff.
Nwosu et al.
B. RT-qPCR showing siMXD1 transfection efficiency and MTT proliferation assay of the HLE cells in complete and Gln-free media.
C. MTT proliferation assay of the indicated inhibitor combinations in HCC cell lines in complete media (CM), 48h.
D. MTT assay of HLE and HLF cell proliferation after treatment with Erlotinib and U0126, 48h.
E. MTT assay of proliferation after treating HUH7 and HLE cells with the indicated combinations of kinase inhibitors, 48h. UT in Figures S7A-E refers untreated group in the complete media or untreated group in the Gln-free media. Statistical significance where applicable is * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Error bars indicate mean±SD.
F. Western blot showing LY294002 inhibition of pAKT in 1h and 24h in HLE cells, and on the right, the effect of the inhibitor in proliferation upon prolonged culture. Cells were pre-treated for 4 days, followed by culture in complete media for 3 days without the inhibitors, and then stained with crystal violet.
Nwosu et al.
Figure S8. Blocking ERK pathway induces metabolic reprogramming
A. Glucose consumption and lactate output after U0126 treatment, 48h. B. Intracellular metabolite levels in HCC cells treated with U0126 in complete media, 24h. C. Glucose carbon labelling of serine, 24h. D. MTT assay, 48h and clonogenic assay after metabolite supplementation in Gln-deprived
HLE cells. Clonogenic assay lasted 7 days followed by 3 days culture in complete media. E. RT-qPCR of transamination genes upon ERK pathway inhibition, 24h. Statistical
significance: * P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
H U H 7
Re
l.mRNA
leve
l0
1
2
G O T 1 M E 1 G P T 1
G lu ta m in eU 0 1 2 6 [2 0 µ M ]E R L O [2 0 µ M ]
---
-+-
-++
---
-+-
-++
---
-+-
-++
*
****
H L E
Re
l.mRNA
leve
l
0 .0
0 .5
1 .0
1 .5
M E 1 G P T 1G O T 1
G lu ta m in eU 0 1 2 6 [2 0 µ M ]E R L O [2 0 µ M ]
---
-+-
-++
---
-+-
-++
---
-+-
-++
***
****
*
***
***
H U H 7
Re
l.mRNA
leve
l
0 .0
0 .5
1 .0
1 .5
2 .0
M E 2 G P T 2G O T 2
G lu ta m in eU 0 1 2 6 [2 0 µ M ]E R L O [2 0 µ M ]
---
-+-
-++
---
-+-
-++
---
-+-
-++
**
H U H 7
Glu
cose
co
nsu
mp
tion
(% o
f U
T)
DM
S O
U0 1 2 6 [1
0 µM]
0
5 0
1 0 0
1 5 0
H U H 7
La
cta
te o
utp
ut
(% o
f U
T)
DM
S O
U0 1 2 6 [1
0 µM]
0
5 0
1 0 0
1 5 0
2 0 0
* * * *
H L ER
el.mRNA
leve
l
0 .0
0 .5
1 .0
1 .5
M E 2 G P T 2G O T 2
G lu ta m in eU 0 1 2 6 [2 0 µ M ]E R L O [2 0 µ M ]
---
-+-
-++
---
-+-
-++
---
-+-
-++
*
**
H L E
D MS O
U 0 1 2 6 [10 µM
]0
5 0
1 0 0
1 5 0 * *
H L E
Re
lativ
e p
rolif
era
tion
(% o
f U
T)
M a la te A s p a r ta te0
5 0
1 0 0
1 5 0
2 0 0 0 m M 1 m M 2 .5 m M 5 m M
*
****
(su p p le m e n ta tio n u p o n G ln w ith d ra w a l)
D.
C.
H L E
D MS O
U 0 1 2 6 [10 µM
]0
5 0
1 0 0
1 5 0
2 0 0 * *
A. B. HUH7
Rel
ativ
e le
vel (
intra
cellu
lar m
etab
.)
PYR
LAC
CIT
aKG
FUM
MAL
SER
MET
GLY
GLN
GLU
ASP
ALA0
100
200CMU0126
****
** ** ** *
****
P=0.
0514
Amino acidsTCA cy.Glycolysis
HLE
Rel
ativ
e le
vel (
intra
cellu
lar m
etab
.)
PYR
LAC
CIT
aKG
FUM
MAL
SER
MET
GLY
GLN
GLU
ASP
ALA0
100
200CMU0126
****
******** *****
**** **
Amino acidsTCA cy.Glycolysis
-Gln
CM
+SER
- +GLU
+ A
SP
HU
H7
S e rin e
13C
-glu
cose
lab
elin
g (%
)
M0
M1
M2
M3
0
5 0
1 0 0
1 5 0 C MU 0 1 2 6
Ñ 1 1 .2 %** * *
S e rin e
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
0
5 0
1 0 0
1 5 0C MU 0 1 2 6
****
Ñ 9 .5 %
S e rin e
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
0
5 0
1 0 0
1 5 0 - G ln- G ln + U 0 1 2 6
S e rin e
13C
-glu
cose
lab
elin
g (
%)
M0
M1
M2
M3
0
5 0
1 0 0
1 5 0- G ln- G ln + U 0 1 2 6
HLE
E.
Nwosu et al.
Figure S9. ERK pathway gene expression in HCC datasets
A. Heatmap showing the expression pattern of growth factors/ERK pathway related genes in 5 human HCC datasets.
B. Heatmap showing the expression of the 24-ERK pathway gene signatures identified in our study and used to stratify HCC into highERK and lowERK groups.
C. Venn diagram depicting overlap of published gene signatures of poor prognosis (PP) to sorafenib in human HCC (Pinyol et al., 2019) and upregulated genes in highERK HCC. # indicates number of genes overlapped with the highERK genes in GSE14520 (# = 10), GSE25097 (# = 15) and both datasets (# = 11).
A. B.
C.XRCC6CAPZA2CHERPEIF4E2CAMK2N1DHX8FLAD1SRP72PSMA1RRP7A
CPSF1RAP1GAPMORC2ATP2A2MAPK14FNDC3BSLC39A1ADARCTBP1CDC42EP1PPIG
NCSTNNME4MRPS21ARHGAP1DDX17NUFIP2PLXNB1AGTBAT3DDIT4TMED9GIT2DDX39LPCAT3SEPT9
#10 #11#15
Nwosu et al.
Table S1. Serine level in human HCC tissues/blood as observed in published metabolomics studies
Sample *Significant change in
Serine level
HCC Patients (N)
Reference
Serum/Plasma Down 82 Chen et al., 2011 Ö Up 30 Fitian et al., 2014 Ö Up 39 Gao et al., 2015 Ö Up **63 Di Poto et al., 2017
Tissue Up 50 Huang et al., 2013 *Reported P-value at least < 0.05 **Compared to cirrhotic controls
Nwosu et al.
Table S2. Expression of serine pathway and NRF2-oxidative stress pathway components in highERK HCC tumours
GSE14520 (high/lowERK) GSE25097 (high/lowERK) logFC P-value Adjusted P logFC P-value Adjusted P
GLDC 0.06 0.7062 0.7600 0.14 0.6532 0.7730 HMOX1 0.20 0.1941 0.2601 0.66 0.0712 0.1639 KEAP1 0.14 0.0679 0.1054 0.09 0.4933 0.6453
NFE2L2 0.20 0.0251 0.0451 -0.16 0.1444 0.2754 NQO1 0.84 0.0033 0.008 2.36 0.0052 0.0206
PHGDH 0.15 0.2741 0.3485 0.19 0.6482 0.7697 PSAT1 0.29 0.062 0.0976 0.17 0.2855 0.4464 PSPH 0.82 7.25E-10 3.14E-08 0.31 1.24E-15 2.85E-13
SHMT1 -0.32 0.0009 0.0027 -0.81 1.31E-13 1.59E-11 SHMT2 0.11 0.2140 0.2827 0.58 0.0804 0.1793
SOD2 0.03 0.8548 0.8847 -1.29 0.1135 0.2311
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Table S3. List of compounds and inhibitors
Compounds/Inhibitors Pathway/Target inhibited
Order number Source
Erlotinib EGFR 5083S Cell Signaling U0126 MEK/ERK 9903S Cell Signaling
Trametinib MEK/ERK 16292 Cayman Chem. SCH772984 ERK 19166 Cayman Chem. PD 0325901 MEK/ERK 13034 Cayman Chem.
Sorafenib Multi-kinase S7397 Selleckchem AOA pan-transaminase 13408-1G Sigma
Asparaginase glutaminolysis A3809 Sigma 10058-F4 c-MYC F3680-5MG Sigma
L-Aspartic acid dimethyl ester - 456233-5G Sigma Serine - S4311-25G Sigma Glycine - G8790-100G Sigma
Methionine - M5308-25G Sigma L-Alanine - A7469-10MG Sigma
Asparagine - A4159-25G Sigma LY294002 PI3K/AKT 1130 Tocris
Dimethyl malate - 374318-5G Sigma Dimethyl 2-oxoglutarate(αKG) - 349631-5G Sigma
EGF - AF-100-15 PeproTech 2-Deoxy-Glucose Glycolysis D6134-1G Sigma L-Glutamic acid - G8415-10MG Sigma
Abbreviations: AOA – Aminoxyacetate; EGF – Epidermal growth factor
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Table S4. RT-qPCR primers
Primer Amplicon Size
Forward Reverse
AREG 97 GTGGTGCTGTCGCTCTTGATA CCCCAGAAAATGGTTCACGCT FASN 188 ACAGCGGGGAATGGGTACT GACTGGTACAACGAGCGGAT
GLS 114 AGGGTCTGTTACCTAGCTTGG ACGTTCGCAATCCTGTAGATTT GOT1 72 CAACTGGGATTGACCCAACT GGAACAGAAACCGGTGCTT GOT2 138 TTACGTTCTGCCTAGCGTCC ACTTCGCTGTTCTCACCCAG
GPT 75 GGTCTTGGCCCTCTGTGTTA TCCGCCCTTTTCTTGGCATC GPT2 103 GACCCCGACAACATCTACCTG TCATCACACCTGTCCGTGACT
HMOX1 155 AAAGTGCAAGATTCTGCCCCC CAGCATGCCTGCATTCACAT ME1 85 TCTTGGCTTGGGAGACCTTG ATTCATCCCTCCGCAAGCTG ME2 177 CGACGGTTGGTCTTGCCTG CCAGATCTCCAAGACCCAGAAT
MPC1 185 CATGAGTACGCACTTCTGGGG GGCATGCAAACAGAAGCCAG MXD1 165 CGTGGAGAGCACGGACTATC CCAAGACACGCCTTGTGACT MYC 119 GGCTCCTGGCAAAAGGTCA CTGCGTAGTTGTGCTGATGT
NFE2L2 81 ATGACAATGAGGTTTCTTCGG CAATGAAGACTGGGCTCTC NQO1 81 ACATCACAGGTAAACTGAAGG TCAGATGGCCTTCTTTATAAGC PPIA 98 GACTGAGTGGTTGGATGGCA TGCCATTCCTGGACCCAAAG
PSAT1 166 ACAGGAGCTTGGTCAGCTAAG CATGCACCGTCTCATTTGCG SOD2 83 TGGTTTCAATAAGGAACGGG GAATAAGGCCTGTTGTTCCT
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Additional Methods
Proliferation and clonogenic assays
2.5 – 7.5 x 103 cells per well were seeded in quadruplicates in 96-well plates and incubated
overnight. The next day, the cells were treated with the indicated compounds. At the end of the
experiment, MTT reagent was added to each well, followed by 3 – 4 h incubation at 37 °C. Media
was then aspirated off and the formed formazan crystals were dissolved with 200 µl of
solubilization reagent (4 parts dimethyl sulfoxide, 4 parts 10% sodium dodecyl sulphate, 2 parts
PBS, and acetic acid at 1.2% of the total reagent volume). Thereafter, the plate was incubated
overnight at 37 °C after which absorbance was read at 560 nm with background correction at 670
nm using Infinite 200 Spectrophotometer (Tecan GmbH, Austria). For clonogenic assay, the cells
were seeded in 6 or 12 well plates, mostly at 1000 – 2500 cells and for the indicated duration.
Thereafter, cells in each well were briefly fixed with methanol followed by staining for ~20 minutes
with 0.5% crystal violet solution diluted 50% v/v with methanol. The plates were then washed in
running tap water, allowed to dry and images obtained using digital camera or scanner.
Viability Assays For trypan blue viability assay, media was aspirated off, followed by 1x wash with HBSS and
trypsinization. The cells were then re-suspended in complete media, mixed 1:1 with 0.4% trypan
blue (Invitrogen) and counted using New Improved Neubauer Chamber. CellTitre Glo viability
assay (Ref #: G9242, Promega) was performed according to manufacturer’s instruction.
Metabolomics profiling of serine-free or -supplemented HLE cells Intracellular metabolite profiling for the experiment on Gln-deprived serine-supplemented HLE
cells and intracellular/extracellular metabolite profiling on the serine-deprived Gln-deprived HLE
cells were performed with Agilent 6470 Triple Quadrupole liquid chromatography tandem-MS (LC-
MS/MS). 3.5 x 105 HLE cells were cultured in 6-well plates in triplicates for 24h and 48h
respectively in DMEM (D9802-1, USBiological) with or without serine/Gln as desired. At the end
of the experiment, the samples were extracted with 80% methanol for lysates (intracellular) and
100% methanol for culture media (extracellular). The methanol-cell suspension was centrifuged
for 10 minutes at 4°C, and the supernatant collected into a new tube after normalization to protein
content of parallel plates. The samples were dried using SpeedVac Concentrator and analysed
by mass spectrometry. The metabolite peaks were integrated using Agilent MassHunter
Quantitative software (version B.09.00).
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ELISA-based protein microarray Quantification of phosphorylated proteins was performed with custom made ELISA-protein
microarrays (ArrayStripTM, Alere Technologies GmbH, Jena, Germany) following the kinetic
detection protocol described previously (Holenya et al., 2011). After glutamine deprivation or
serine supplementation, the cells were lysed with 6 M Urea lysis buffer containing
phosphatase/protease inhibitors. Total protein concentration was determined with Pierce BCA
Protein Assay kit (Thermo Fischer) and 1 µg of total protein was loaded on microarrays for each
sample. The results were acquired using an Arraymate reader (Alere Technologies GmbH, Jena,
Germany).
Glucose, lactate and oxygen consumption assays 1.5 – 2 x 105 HUH7 and HLE cells were cultured overnight in triplicates in 12 well plates. Media
was changed to that containing treatment conditions and the cells were further cultured for 48 h.
Media glucose/lactate were measured using Roche Cobas C311 Chemistry Analyzer according
to the manufacturer’s instruction. The metabolite levels were normalized to protein content or
absolute cell count and the experiments were repeated at least twice. For O2 measurement, HUH7
and HLE cells were seeded in 24-well OxoDish plate (PreSens Precision Sensing GmbH,
Germany), and cultured overnight in complete media. Next day, the media was removed, followed
by a 1x wash of the adherent cells with HBSS. Complete or Gln-free media was introduced, and
the cells incubated further. Measurements of consumed oxygen were taken automatically every
10 minutes for 72h. Thereafter, fresh complete media was re-introduced, and the cells incubated
for another 48h with O2 measurement every 10 minutes. Analyses of the acquired data were
performed as previously described (Lochead et al., 2015).
siRNA transfection Smartpool: siGENOME siNFE2L2 (NRF2) and siMXD1 (5 nmol) were purchased from Dharmacon
(Life Technologies) and reconstituted to a 10 µM stock concentration. Transfection was done using
Lipofectamine RNAiMax reagent (Invitrogen, USA) as per manufacturer’s instruction.
Subsequently, the cells were incubated for 24 – 48 h before sample collection or further
experiment.
Western blotting For detection of total ERK after pERK, stripping was applied. For this, Restore™ Western Blot
Stripping Buffer (#21059 Thermo Scientific) was used according to manufacturer’s instruction.
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Alternatively, stripping was done with a recipe prepared following the protocol from Abcam Plc
(https://www.abcam.com/protocols/western-blot-membrane-stripping-for-restaining-protocol).
Isolation and culture of primary mouse hepatocyte Primary hepatocytes used in this study were freshly isolated from male C57BL/6 wild type mice
as previously described (Dropmann et al., 2016). Following isolation, hepatocytes were seeded in
culture plate and incubated 4 h to overnight in Williams Media (10% FBS, 1% P/S, 1% L-Gln +
0,1% Dexamethasone) after which they were cultured for 48h with DMEM under the indicated
experiment conditions.
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