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

Nwosu et al.

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

Nwosu et al.

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).

Nwosu et al.

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.

Nwosu et al.

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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