Supplementary Fig. S1

Supplementary Fig. S1

Supplementary Figure S1. The co-expression profile among ALDH activity, memGrp78 and Glut3 in SAS Sphere cells and cisplatin-resistant (cisPtR) SAS cells was examined by FACS. (A) The co-expression profile among ALDH, CD133 and Glut3 in SAS Sphere cells and cisplatin-resistant (cisPtR) SAS cells was examined by FACS. Results are means ± SD of triplicate samples from three experiments (***, p < 0.001) (B).

A

ALD

H+

cel

ls (

%)

SAS-S

memGrp78(%)

Glu

t3(%

)

SAS cisPtR

memGrp78(%)

Glu

t3(%

)

B SAS-S

CD133(%)

Glu

t3(%

)

SAS cisPtR

CD133(%)

Glu

t3(%

)

G1 G2

G3G4

G1 G2

G3G4

G1:

Glu

t3+ m

emG

rp78

-

SAS cisPtRSAS-S

35.4%

60.1%

20.2%

10.2%

24.9%

62.6%

25.2%

5.24%

G1 G2

G3G4

G1 G2

G3G4

SAS cisPtRSAS-S

34.1%

62.9%

22.8%

11.8%

33.5%

59.8%

33.6%

6.44%

G1G2G3G4

010203040506070

*********

*********

SAS cisPtRSAS-S0

10203040506070

************

******

SAS cisPtRSAS-S

G1G2G3G4

ALD

H+

cel

ls (

%)

ALDH(%) ALDH(%)

G2:

Glu

t3+ m

emG

rp78

+

G3:

Glu

t3- m

emG

rp78

+

G4:

Glu

t3- m

emG

rp78

-

G1:

Glu

t3+ C

D13

3-

G2:

Glu

t3+ C

D13

3+

G3:

Glu

t3- C

D13

3+

G4:

Glu

t3- C

D13

3-


• Supplementary Figure S2. Single-cell suspension from parental SAS or sphere SAS cells was stained with (A) CellROX Deep Red reagent and (B) DCF (oxidation-insensitive analog). Then, the intracellular level of ROS in parental SAS or sphere SAS cells was determined by FACS analyses.

Supplementary data

A

CellROX Deep Red

Co

un

t

SAS-P SAS-S

15.7%

SAS-P

SAS-S

Co

un

t

15.7%4.3%

CellROX Deep Red

0.7%

Co

un

t DCF+

B

DCF+

Co

un

t

SAS-P SAS-S

0.7%0.3%


Supplementary Figure S3. Enrichment of the drug-resistant population after in vitro drug treatment. Image of enrichment of the cisplatin-resistant population cells. Treatment of 5µM cisplatin killed more than 50% of cells at 48 h. The majority of cells died by the fifth day. Over the next 20 days few enlarged cells were seen with flattened, senescent-like morphology. Then the supernatant were replaced with fresh medium containing drugs at 48hr. After two more cycles, sphere cells were seen. cisPt, cisplatin.

Supplementary data

cis-PtR cisPt 10 µM 72hr Recover 20 days

cisPt 10 µM 72hrSAS-P Recover 20 days Recover 30 days

A


Supplementary data

A

control H2O2 Arsenic

ROSLow

B SAS-S

RO

SL

ow

RO

SM

ed

iR

OS

Hig

h

0102030405060

% S

pher

e-fo

rmat

ion

effic

ienc

y

ROSLow

ROSMedi

ROSHigh

******

Supplementary Figure S4. ROSLow cells were sorted using DCFDA staining from SAS sphere cells. At day 21, the cells were treated with H2O2 or arsenic,respectively, for 72 hours. Representative images of chemical induced differentiation were shown. (A) A single cell of ROSLow, ROSMedi and ROSHigh under defined serum-free selection medium was plated in each well of 96-well low attachment plates, respectively. After 14 days, counted the number of spheres and calculated the sphere-forming efficiency (B).


Supplementary Figure S5. Differentially expressed genes of reactive oxygen species scavenging in Parental cells and Sphere cells under 2, 3, 5, or 9 weeks of cultivation with defined serum-free selection medium were collected and analyzed. (A) The heat maps of some ROS scavenger genes in parental versus sphere cells. Red and blue indicate high and low expression levels, respectively. SAS-P, SAS parental; OECM1-P, OECM1 parental; SAS-S, SAS Sphere; OECM1-S, OECM1 Sphere; CAT, catalase; PRDX3, peroxiredoxin 3; SOD2, superoxide dismutase 2.

Supplementary data

A

OECM1-P

SOD2

CAT

PRDX3

SAS-P SAS-SphereOECM1-Sphere

Paren

tal

3 wee

ks

5 wee

ks

9 wee

ks

Paren

tal

3 wee

ks

5 wee

ks

9 wee

ks

B


Supplementary data

Supplementary Figure S6. Treatment of sphere cells with catalase inhibitor 3AT reduced catalase activity. (A) SAS-S cells or (B) OECM1-S cells were pretreated with catalase inhibitor (3AT), for 72hr, afterward, we measured the activities of catalase as described in Materials and Methods.

A

SAS-PSAS-SSAS-S 3AT

Cat

ala

se s

pec

ific

act

ivity

0.020.040.060.080.10.120.140.160.18

00

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Cat

ala

se s

pec

ific

act

ivity

OECM1-POECM1-SOECM1-S 3AT

5.7%7.6%

6.8%8.3%

1.5%

21.8%13.0%

SAS-SControl cisPt

2ME 2ME +cisPt

3AT + cisPt3AT

3AT + 2ME + cisPt

Co

un

t DCFDA

B

6.4% 18.2%

7.0%

68.5%

5.6 12.8%

5.6%

76.0%

SAS-SControl cisPt

2ME 2ME + cisPt

3AT + cisPt3AT

3AT + 2ME + cisPt10.0% 42.9%

11.9%

35.2%

3.5% 8.1%

4.3%

84.1%

4.2% 14.9%

7.0%

74.0%

5.6% 20.8%

13.1%

60.5%

5.3% 17.3%

9.7%

67.6%

Pro

pid

ium

io

did

e(P

I)+

AnnexinV


Supplementary data

Supplementary Figure S7. Combinatorial treatment of ROS scavenger inhibitor and cisplatin induces apoptosis cell death. (A) SAS-Sphere cells were either singly treated with cisplatin or co-treated with ROS scavenger (2ME and 3AT), for 72hr, afterward, and stained with DCFDA plus (B) Annexin V/Propidium iodide (PI); then, examined by flow cytometry. (C) The amount of CK18 positive cells was determined by staining the drugs treated cells with CK18 antibody, and then examined by flow cytometry (2ME: superoxide dismutase 2; 3AT: 3-Amino-1,2,4-triazole).

A

Co

un

t

56.3% 2ME+3AT+cisPt

ControlCisPt

CK18+ cells (%)

C

B


Supplementary data

A

CD44(%)

45.9% 28.0% 33.5%

25.7% 32.9%

shLuc

shSOD2#1 shSOD2#2

shCAT#1 shCAT#2

Co

un

t

C

D

Sph

ere

No

shLu

csh

SO

D2

shC

AT

SAS-S

02468101214161820

SAS-S

shLucshCAT#1shCAT#2shSOD2#1shSOD2#2

0510152025303540

SAS-S


05101520253035404550

SAS-S


RO

SL

ow c

ells

(%

)

Ck1

8 ce

lls (

%)

CD

44 c

ells

(%

)

Col

ony

No

shLu

csh

SO

D2

shC

AT

SAS-S

0

10

20

30

40

50

60

70

shLucshCATshSOD2

0

5

10

15

20

25

30

shLucshCATshSOD2

Supplementary Figure S8. Knockdown of CAT or SOD2 gene expression diminished spheres-forming capability, stemness marker expression of HN-CICs. The percentages of (A) ROS- cells, (B) CK18+ cells and (C) CD44+ cells in shCAT, shSOD2 and vector control SAS sphere cells were compared by flow cytometry analysis, respectively. SAS sphere cells were first infected with sh-CAT-1, sh-CAT-2, sh-SOD2-1, sh-SOD2-2 or sh-Luc lentivirus, and further cultivated under defined serum-free selection medium. The sphere formation capability (D) and anchorage independent growth ability (E) of SAS sphere cells treated with either sh-Luc or CAT or SOD2-shRNA lentivirus were examined by microscope.

E


Supplementary data

22.3%16.8%

PEG-Control PEG-CAT

memGrp78(%)

SAS-S

SS

C

16.3%

PEG-Control PEG-CAT

ROS(%)

SAS-S

SS

C

24.6%

A

C

0

5

10

15

20

25

30

SAS-S

PEG-ControlPEG-CAT***

RO

SL

ow c

ells

(%

)

***

0

5

10

15

20

25

SAS-S

me

mG

rp78

cel

ls (

%)

PEG-ControlPEG-CAT

29.6%

OECM1-S 3AT

43.8%

OECM1-S

54.8%

OECM1-S PEG-CAT

47.8%

SAS-S

60.6%

SAS-S PEG-CAT

41.9%

SAS-S 3AT

Co

un

t

D

CD44(%)

Supplementary Figure S9. Overexpression of CAT in HNCICs promotes stemness properties. (A) SAS-S cells or OECM1-S cells were treated with PEG-CAT (100 U/ml) , for 72hr, afterward, the intracellular catalase activities were measured . The percentages of (B) ROS- cells, (C) memGrp78+ cells and (D) CD44+ cells in treated with PEG-CAT (100 U/ml) or control SAS sphere cells were analyzed by flow cytometry, respectively. (***, p<0.001)

00.050.10.150.20.250.30.350.40.45

***

*

B

Cat

ala

se s

pec

ific

act

ivity

SAS-S OECM1-S

PEG-ControlPEG-CAT


Supplementary data

A

OECM1-P PEG-CAT

37.8%17.2%

30.3%12.0%

OECM1-P PEG-Control

SAS-P PEG-Control SAS-P PEG-CAT

Co

un

t

ALDH(%)05

1015202530354045

SAS-P OECM1-P

PEG-ControlPEG-CAT

ALD

H c

ells

(%

)

***

***

Supplementary Figure S10. Overexpression of CAT in HNSCC under defined serum-free selection medium promotes stemness properties. The activity of ALDH in SAS cells treated with PEG-CAT (100 U/ml) or control PEG under further cultivation within defined serum-free medium for 4 days was analyzed by flow cytometry.(***, p<0.001)


Supplementary data

Supplementary Figure S11. Cell viability of drugs treated hematopoietic stem cells. (A) Hematopoietic stem cells (HSC) were treated with 5 µM cisplatin, 15 mM 2ME or 25 µM 3AT, respectively, for 72 hours. Cell viability of the treated cells was further determined by MTT assay. HSC, Hematopoietic stem cells.

A

Cel

l Via

bilit

y (%

)

***

0

20

40

60

80

100

120Control

cisPt 5 µM

HSC

***

0

20

40

60

80

100

120Control

2ME 15 µM

HSC

***

0

20

40

60

80

100

120Control

3AT 25 mM

HSC

Cel

l Via

bilit

y (%

)

Cel

l Via

bilit

y (%

)

Documents

Supplementary Fig. S1