5
- KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 He p3B HuH-7 0 5 10 15 20 25 Percentage of SV mRNA / KLF6 SV1 SV2 SV3 Supplementary Figures A C B IHH-KLF 6 IHH-SV2 IHH-GF P p53 mRNA expression (2exp (-Ct) ) 0.5 1.0 1.5 2.0 2.5 0 HepG2-S V 2 HepG2 p53 mRNA expression (2exp (-Ct) ) 0.5 1.0 1.5 2.0 2.5 0 MDM 2 75 kDa- 50 kDa- -Actin IH H-GFP IHH-KL F 6 IHH-SV2 37 kDa- 105 kDa- HepG2 HepG2- S V 2 MDM2 -Actin 75 kDa- 105 kDa- 50 kDa- 37 kDa-

- KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 Hep3B HuH-7 0 5 10 15 20 25 Percentage of

Embed Size (px)

Citation preview

Page 1: - KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 Hep3B HuH-7 0 5 10 15 20 25 Percentage of

- KLF6 869 bp

IHH

Hep

G2

Hep

3B

HuH

-7

900 bp-800 bp-700 bp-

600 bp- SV1 715 bp

SV2 743 bp SV3 745 bp

IHH

Hep

G2

Hep

3B

HuH

-7

0

5

10

15

20

25

Per

cent

age

of S

V m

RN

A /

KLF

6

SV1SV2SV3

Supplementary Figures

A

C

B

IHH

-KLF

6

IHH

-SV2

IHH

-GFP

p53

mR

NA

exp

ress

ion

(2e

xp(-

Ct))

0.5

1.0

1.5

2.0

2.5

0

Hep

G2-

SV2

Hep

G2

p53

mR

NA

exp

ress

ion

(2e

xp(-

Ct))

0.5

1.0

1.5

2.0

2.5

0

MDM 275 kDa-

50 kDa--Actin

IHH

-GFP

IHH

-KLF

6IH

H-S

V2

37 kDa-

105 kDa-

Hep

G2

Hep

G2-

SV2

MDM2

-Actin

75 kDa-

105 kDa-

50 kDa-37 kDa-

Page 2: - KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 Hep3B HuH-7 0 5 10 15 20 25 Percentage of

D

0 24h 48h 72hTime

Cel

l pro

lifer

atio

n Hep3B

Hep3B-SV2

02

04

06

08

01

00

Co

unts

0 200 400 600 800 1000

Propidium Iodide

Hep3B-SV2

0 200 400 600 800 1000

Propidium Iodide

02

04

06

08

01

00

Co

unts

Hep3B

Sub-G1

GO/G1

G2/M

S

Sub-G1

GO/G1

G2/M

S

Sub-G1 GO/G1 S G2/M

Hep3B

Hep3B-SV2

Per

cent

age

of c

ells

0

10

20

30

40

50

60

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

E

Page 3: - KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 Hep3B HuH-7 0 5 10 15 20 25 Percentage of

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 23 24 25 26 27 28 29

F

SV

2 / K

LF6

ratio

in H

CC

com

pare

d to

ST

Normal(n=5)

Cirr(n=28)

HCC (n=30)

KLF

6 m

RN

A e

xpre

ssio

n(2

exp(-

Ct))

N.S.

0

0.5

1.0

1.5

2.0

2.5

3.0

G

N.S.

N.S.

H

00.20.40.60.81.0

1.21.41.61.82.0

10 15 20 18 21 14 6 23 9 24 19 28 11 3 8 2 13 1 25 26 4 7 12 22 16 27 5 29

3.05.07.09.0

11.013.015.017.0

KLF

6 m

RN

A e

xpre

ssio

n (f

old

chan

ge in

HC

C vs

surr

ound

ing

tissu

e)

Page 4: - KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 Hep3B HuH-7 0 5 10 15 20 25 Percentage of

0

1

2

3

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 22 23 24 25 26 27 28 29

I

SV

1 / K

LF6

ratio

in H

CC

com

pare

d to

ST

SV

1 m

RN

A e

xpre

ssio

n(2

exp(-

Ct))

**

N.S.

0

0.05

0.10

0.15

0.20

0.25

0.30

Normal(n=5)

Cirr(n=28)

HCC(n=30)

J

IHH

-KLF

6

IHH

-SV2

IHH

-GFP

SV

1 m

RN

A e

xpre

ssio

n (

2exp

(-

Ct))

0.5

1.0

1.5

2.0

2.5

0

Hep

G2-

SV2

Hep

G2

N.S.

N.S.

N.S.

IHH

-KLF

6

IHH

-SV2

IHH

-GFP

SV

3 m

RN

A e

xpre

ssio

n (

2exp

(-

Ct))

0.5

1.0

1.5

2.0

2.5

0

Hep

G2-

SV2

Hep

G2

N.S.

N.S. N.S.

Page 5: - KLF6 869 bp IHH HepG2 Hep3B HuH-7 900 bp- 800 bp- 700 bp- 600 bp- SV1 715 bp SV2 743 bp SV3 745 bp IHH HepG2 Hep3B HuH-7 0 5 10 15 20 25 Percentage of

Supplementary figures.A- Qualitative PCR for KLF6 and its splice variants in different hepatic cell lines using primers that permit amplifications of KLF6 and SV variants. PCR products were visualized after agarose gel electrophoresis and ethidium bromide staining. The graph represents the expression of KLF6 splice variants determined by semi-quantitative RT-PCR. Results are expressed as a percentage of KLF6 mRNA expression. The results represent the mean of three different amplifications. B- Expression of p53 mRNA in IHH (left graph) and HepG2 (right graph) stable cell lines by qRT-PCR. Results represent the mean of three amplifications and are calculated using the 2exp(-Ct) formula. p53 mRNA level in IHH-GFP and HepG2 cells is used as control. C- Expression MDM2 protein in IHH (left panel) and HepG2 (right panel) stable cell lines by Western blot. The gels are representative of three analyses D- Proliferation of Hep3B and Hep3B-SV2 cell lines. After adherence, cells were counted (time 0) and then at every 24h interval until 72h. The graph represents the cell population growth compared to the population at time 0 (expressed as 1). The results represent the mean of three experiments. E- Percentage of cells in the different phases of the cell cycle measured by flow cytometry. Each panel is representative of three analyses. The graph represents the average percentage of cells in the different phases of the cell cycle obtained from three analyses. F- Representation of SV2 expression relatively to KLF6 expression in HCC vs ST in 28 patients. Results are expressed as the ratio SV2/KLF6 expression in HCC vs ST.G- Overall KLF6 mRNA level in normal liver (Normal n=5), surrounding cirrhotic tissues (Cirr n=28) and HCC samples (n=30) measured by qRT-PCR. Results are the mean of three different amplifications and are expressed as 2exp –Ct. N.S. stands for Not Significant. H- KLF6 mRNA expression in 28 patients measured by qRT-PCR. For each patients, KLF6 mRNA level in the tumor is represented as fold change compared to the ST. The values represent the mean (±SEM) of three different quantifications performed in triplicate. For technical reasons, results for samples #17 and #30 could not be obtained. I- Top graph: Representation of SV1 expression relatively to KLF6 expression in HCC vs ST in 28 patients. Results are expressed as the ratio SV1/KLF6 expression in HCC vs ST. Bottom graph: Overall SV1 mRNA level in normal liver (Normal n=5), surrounding cirrhotic tissues (Cirr n=28) and HCC samples (n=30) measured by qRT-PCR. Results are the mean of three different amplifications and are expressed as 2exp –Ct. * p < 0.05. J- Expression of the splice variants SV1 and SV3 in response to SV2 cDNA over-expression by qRT-PCR. SV1 and SV3 expression was measured in IHH-SV2 (left panel) and HepG2-SV2 (right panel) and compared to its level in IHH-GFP, HepG2-GFP and Hep3B cell lines, respectively. Results are the mean of three different amplification and are calculated using the 2exp(-DDct) formula. SV2 mRNA level in IHH-GFP and HepG2-GFP is used as control. N.S. stands for Not Significant..