Description of Supplementary Files

File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables

Supplementary figure 1. The protein expression of ENTPD2 in HCC cell lines and tissues. (a)

The protein expressions of ENTPD2 in MHCC97L, Hep3B and PLC/PRF/5 exposed to 20% and 1%

O2 for 48 hr were determined by flow cytometry (n = 3). (b) Immunohistochemical staining of

ENTPD2 in human HCC tissues displayed a patchy pattern which is a typical oxygen diffusion

pattern. Data are presented as mean±s.d. (Student’s t-test, *** P<0.001)

MHCC97L

Hep3B

PLC/PRF/5

0.0

0.5

1.0

1.5

2.0 20% O2 1% O2

ENTP

D2

prot

ein/

Med

ian

Fluo

resc

ence

Inte

nsity

MHCC97L Hep3B PLC/PRF/5

Cou

nt

ENTPD2 fluorescence intensity

a

b

50 µm 100 µm

****** ***

Supplementary figure 2. The knockdown efficiency of HIF-1α and HIF-2β in MHCC97L

confirmed by western blot. HIF-1α and HIF-2α protein expressions in MHCC97L-EV, -shHIF-1α, -

shHIF-2α cells that were exposed to 20% and 1% O2 for 4 hours.

Supplementary figure 3. The mRNA expression of CD39 family in ENTPD2 knockdown HCC

cells. The mRNA expressions of ENTPD1, ENTPD2, ENTPD3 and ENTPD8 in MHCC97L-NTC and

–shENTPD2 clones. Cell were exposed to 20% and 1% O2 for 24 hr (n = 3). qRT-PCR values were

normalized to 20% O2. Data are presented as mean±s.d.

ENTPD1 ENTPD2 ENTPD3 ENTPD80

5

10

15

2020% O2 NTC20% O2 shENTPD2_120% O2 shENTPD2_21% O2 NTC1% O2 shENTPD2_11% O2 shENTPD2_2

ENTP

D F

amily

mR

NA/

18S

Supplementary figure 4. Proliferation rate of Hepa1-6-EV and -shEntpd2 clones (n = 3). Data

are presented as mean±s.d. No statistically significant difference was found between the proliferation

of Hepa1-6-EV and –shEntpd2 clones. (Student’s t-test, P>0.05)

0 24 48 72 960

10

20

30

40 EV shEntpd2_1Hepa1-6

shEnptd2_2

Hours

Cel

l Num

ber (

105 )

Supplementary figure 5. The mRNA expression of Entpd2 in mouse HCC cell lines. The mRNA

expressions of Entpd2 in Hepa1-6 and MM189. Cells were exposed to 20% and 1% O2 for 24-48 hr (n

= 3). qRT-PCR values were normalized to 20% O2. Data are presented as mean±s.d. (Student’s t-test,

* P<0.05, ** P<0.01)

Hepa1-6

24hr

48hr

0.0

0.5

1.0

1.5

2.0

2.5 20% O21% O2

Entp

d2 m

RNA

MM189

24hr

48hr

0

2

4

6 20% O21% O2

Entp

d2 m

RNA

*

**

**

**

Supplementary figure 6. Establishment of MHCC97L-sgENTPD1/2 and Hepa1-6-sgEntpd1/2

clones. Protein expressions of ENTPD1/2 in MHCC97L and Entpd1/2 in Hepa1-6 clones were

determined by flow cytometry (n = 3). Grey area indicated the control isotype.

Enptd1

Enptd2ENTPD2

ENTPD1

Hepa1-6

Hepa1-6

MHCC97L

MHCC97L

EVsgENTPD1_1sgENTPD1_2

EVsgENTPD2_1sgENTPD2_2

EVsgEntpd1_1sgEntpd1_2

EVsgEntpd2_1sgEntpd2_2

Supplementary figure 7. The effects of ENTPD1 and ENTPD2 on HCC growth. C57BL/6 mice

were orthotopically implanted with 3×106 Hepa1-6-EV, -sgEntpd1 and -sgEntpd2 (n = 6 for each

group). Tumor size was measured with caliper. (Student’s t-test, *** P<0.001)

2cm

EV

sgEntpd1

sgEntpd2

Hepa1-6-syngeneic HCC model

EV

sgEntp

d1

sgEntp

d20

1

2

3

4

5 n.s.***

Tum

or s

ize´

103

(mm

3 )

Supplementary figure 8. Establishment of MHCC97L-ENTPD2 overexpressing (OE) clones. (a)

mRNA and (b) protein expressions of ENTPD2 in MHCC97L-EV and -ENTPD2-OE clones were

determined by qRT-PCR and flow cytometry respectively (n = 3). Grey area indicated the control

isotype. The expressions were normalized to EV. Data are presented as mean±s.d. (Student’s t-test,

*** P<0.001)

EV

ENTPD2_OE

0

100

200

300

400

***

ENTP

D2

mR

NAEVENTPD2_OE

EV

ENTPD2_OE

0

1000

2000

3000

4000 ***

ENTP

D2

prot

ein/

Med

ian

Fluo

resc

ence

Inte

nsitya b

ENTPD2

Count

Supplementary figure 9. The effects of ENTPD2-knockout on extracellular ATP hydrolysis and

5’-AMP production in HCC cell lines. (a) MHCC97L and (b) Hepa1-6 cells were pre-exposed to

20%O2 and 1%O2 for 48 hr. Cells were trypsinized and 2×106 cells were re-suspended in serum-free

DMEM medium supplemented with 100 µM ATP and incubated at 37 oC for 1 hr. CM were collected

and subjected to LC-MS analysis (n = 3). (c) Representative peaks of 5’-AMP in 5’-AMP standard

solution, and CM from MHCC97L cells exposed to 20%O2 and 1%O2. Data are presented as

mean±s.d. (Student’s t-test, * P<0.05, ** P<0.01, *** P<0.001)

MHCC97L

EV

sgENTPD2_

1

sgENTPD2_

2 EV

sgENTPD2_

1

sgENTPD2_

20

1

2

3

4

5 20% O2 1% O2

*****

Rel

ative

rate

of A

TP h

ydro

lysis MHCC97L

EV

sgENTPD2_

1

sgENTPD2_

2 EV

sgENTPD2_

1

sgENTPD2_

20

1

2

3

4

5 20% O2 1% O2

********

Rel

ative

5'-A

MP

leve

l

Hepa1-6

EV

sgEntp

d2_1

sgEntp

d2_2 EV

sgEntp

d2_1

sgEntp

d2_2

0

2

4

6

8 20% O2 1% O2

*****

Rel

ative

rate

of A

TP h

ydro

lysis Hepa1-6

EV

sgEntp

d2_1

sgEntp

d2_2 EV

sgEntp

d2_1

sgEntp

d2_2

0

2

4

6

8 20% O2 1% O2

*********

Rel

ative

5'-A

MP

leve

l

a b

Inte

nsity

5’-AMP

Time, min

5’-AMP standard

20% CM(MHCC97L)

1% CM(MHCC97L)

c

400

150

Supplementary figure 10. The effect of 5’-AMP on total MDSC maintenance. Splenic

CD11b+Gr1+ cells isolated from BALB/c nude mice were cultured in the presence of different

concentrations of 5’-AMP. The percentages of CD11b+Gr1+ cells after 4-day culturing were analyzed

(n = 3). Data are presented as mean±s.d. (Student’s t-test, *** P<0.001)

Gr1

CD11b

0 1 10 100

5’-AMP (µM)

28.3% 28.7% 33.0% 44.8%

0 1 10 1000

10

20

30

40

50 ***

5'-AMP (µM)

BALB

/c N

ude

sple

nic

CD

11b+ G

r1+ c

ells

(%)

Supplementary figure 11. The level of 5’-AMP in MDSC culture. The effect of 5’-AMP on MDSC

maintenance. Splenic CD11b+Gr1+ cells isolated from C57BL/6 mice were cultured in the presence of

100 µM 5’-AMP with or without NT5E inhibitor, APCP. CM were collected after 24 hr and subjected

to LC-MS analysis (n = 3). The peaks representing 5’-AMP and adenosine (ADO) in 5’-AMP/ADO

standard solution and CM were shown.

ADO Standard

MOCK + 5’AMP

APCP + 5’AMP

ADOIn

tens

ity

Time, min

5’-AMP Standard

MOCK + 5’AMP

APCP + 5’AMP

5’-AMP

Inte

nsity

Time, min

Supplementary figure 12. The effect of 5’-AMP on the apoptotic rate of total MDSCs. Splenic

CD11b+Gr1+ cells isolated from C57BL/6 mice were cultured with or without 100 µM 5’-AMP.

Annexin V and propidium iodide (PI) staining was performed in cells at different time points.

Percentage of apoptotic cells was measured by flow cytometry analysis (n = 3). Data are presented as

mean±s.d. No statistically significant difference was found. (Student’s t-test, P>0.05)

24 48 72 96

Hour

100

µM

5’-A

MP

MO

CK

C57BL/6 splenic CD11b+Gr+ cells

0 24 48 72 960

20

40

60

80

100 MOCK100µM 5'-AMP

hour

Apop

totic

cel

l/ %

C57BL/6 splenic CD11b+Gr1+ cells

0 24 48 72 960

2

4

6

8

10 MOCK100µM 5'-AMP

hour

Cel

l cou

nt (1

05 )

PI

Annexin V

Supplementary figure 13. The effect of 5’-AMP on total MDSC differentiation. Splenic

CD11b+Gr1+ cells isolated from (a) C57BL/6 mice or (b) BALB/c nude mice were cultured with or

without 100 µM 5’-AMP for 4 days. The distribution of dendritic cell and total MDSC populations

were determined by the surface marker CD11c by flow cytometry (n = 3). Grey area indicated the

control isotype. (c) The effect of ENTPD2 on the fate of total MDSCs in vivo. Splenic CD11b+Gr1+

cells isolated from C57BL/6 mice were stained with fluorescent cell tracking dye CFSE and co-

inoculated with Hepa1-6-EV or shEntpd2 clones into C57BL/6 mice subcutaneously (n = 3 for each

groups). The tumors were dissociated and stained with anti-CD11c and anti-F4/80 to examine the

differentiation of CFSE-labelled total MDSCs. Data are presented as mean±s.d. (Student’s t-test, *

P<0.05, *** P<0.001)

27.6% 16.3%

MOCK 100 µM 5’-AMP

F4/

80

CD11c

a

CFSE

CFSE

FSC

FSC

CD11c

CD11c

F4/8

0F4

/80

EV

shE

ntpd

2

CFSE-labelled total MDSCs

CD11c+F4/80+/-

dendritic cellsGating::

EV

shEntp

d20

10

20

30

40

*

CFS

E-la

belle

dC

D11

c+ F4/8

0+/- c

ells

/ %

cMOCK

5'-AMP

0

20

40

60

80

100MC

DC

MDSC

DC

MC

MDSC

DC DC

C57

BL/6

spl

enic

CD

11b+ G

r1+ c

ells

(%)

b

CD11c+

MOCK100 µM5’-AMP

MOCK

5'-AMP

0

10

20

30

40

50

***

MD

SC-d

erive

dC

D11

c+ cells

(%)

BALB/c Nude splenic CD11b+Gr1+ cells

Supplementary figure 14. The effect of POM-1 on Hepa1-6-sgEntpd1-derived tumors. C57BL/6

mice were orthotopically implanted with 3×106 Hepa1-6-sgEntpd1. On day 3, mice were administered

with vehicle or 10 mg/kg POM-1 through i.p. injection for 8 consecutive days (n = 7 for each group).

Images of tumors harvested from mice and tumor size was measured with caliper. (Student’s t-test, **

P<0.01)

Vehicle

POM-10

2

4

6

8

**

Tum

or s

ize (1

02 mm

3 )

2 cm

Vehicle

POM-1

Hepa1-6-sgEntpd1-sygeneic HCC model

Supplementary Table 1 | Target sequences of shRNAs and sgRNAs

sh/sgRNA clones Gene Bank Target nucleotides

Human shENTPD2 NM_001246 150-170

Mouse shEntpd2_1 NM_009849 529-549

Mouse shEntpd2_2 NM_009849 629-649

Human shHIF-1α NM_181054 2,123-2,141

Human shHIF-2α NM_001430 1,992-2,012

Human sg-ENTPD2

(activation)

NM_001246 CGGCGGAAGCTTGGGAGCGG

Human sg-ENTPD2_1 NM_001246 GCGTCCCCACCCGCGACGTC

Human sg-ENTPD2_2 NM_001246 GCTCCCGGACGTCGCGGGTG

Human sg-ENTPD1_1 NM_001776 GTTGATAGTAATCCAGCCAT

Human sg-ENTPD1_2 NM_001776 CTCTTGGCCAGTAATGATCC

Mouse sg-Entpd2_1 NM_009849 CGGGCGGCTCCCGGACGTCT

Mouse sg-Entpd2_2 NM_009849 GGCTCCCGGACGTCTTGGGT

Mouse sg-Entpd1_1 NM_001304721 GAGCTATCACAGCCAAGATAG

Mouse sg-Entpd1_2 NM_001304721 CCTTGGTTTCACCTCTATCT

Supplementary Table 2 | Primer sequences

Genes Sequences (5’-3’)

Human ENTPD1 Forward: CAGGGGGATTTTGGGGCATT

Reverse: TACTTCTCCTTTACTCCAGCGT

Human ENTPD2 Forward: ACCCACAGCTTCCTCTGCTA

Reverse: CTGAAGAGCCCAGAAACCAG

Human ENTPD3 Forward: CCATTTGTGGCTTTTGCAGGA

Reverse: CTGACTCCAATTCTGTGAGCA

Human ENTPD4 Forward: AGCGAAGCCATTGTCCGTAA

Reverse: ACTTCTTCCTGCTGTGAGGAC

Human ENTPD5 Forward: GACAGCACAGTCTTACAGCTCA

Reverse: CAATGGGAGATGCCCAGAGAC

Human ENTPD6 Forward: TTCGAGATCGCAGCCAAGTA

Reverse: CCGAGTGAGCTTCAGCACTT

Human ENTPD7 Forward: ATGCAGAACAGCAGGATCAGT

Reverse: AGAAGAGTCCCAGGAATGCCA

Human ENTPD8 Forward: CACAGTTGAAGGGACAGGCA

Reverse: GCTGGCCTCCACATAGAACT

Human NT5E Forward: ATGAACGCAACAATGGCACAA

Reverse: CCTTCTTCAGGGTGGAACCTT

Human 18S Forward: GAGGATGAGGTGGAACGTGT

Reverse: AGAAGTGACGCAGCCCTCTA

Mouse Entpd2 Forward: GCCCTCAAGTATGGCATCGT

Reverse: CGAACATCGCAAGAGCTGTG

Mouse 18s Forward: ACATCGACCTCACCAAGAGG

Reverse: TCCCATCCTTCACATCCTTC

Human ENTPD2-317

(ChIP)

Forward: CTCCACAAACAGGTGCTGAC

Reverse: CGTGGAGGCAGGAGACCT

Human ENTPD2-2215 (ChIP)

Forward: CCACAACCGGCTAATTTTTG

Reverse: TGCCTCAGTCTCCACATCAG

Human ENTPD2-2497 (ChIP)

Forward: GGGTTCACACCATTCTCCTG

Reverse: CGGTGGCTCACTCCTGTAAT