HCT116-TP53KO
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HCT116-TP53KO. Sh MAPK 14. Sh MAPK 14bis. Sh MAPK 12. Sh MAPK 13. Sh MAPK 11. EV. EV. EV. EV. p38. GAPDH. 2.5. 2.0. 1.5. IC50 (nM). 1.0. 0.5. 0.0. Sh MAPK 14. Sh MAPK 14bis. Sh MAPK 11. Sh MAPK 12. Sh MAPK 13. Sh LUC. HCT116-TP53KO. HCT116-TP53KO.

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HCT116-TP53KO

Sh

MAPK

14

Sh

MAPK

14bis

Sh

MAPK

12

Sh

MAPK

13

Sh

MAPK

11

EV

EV

EV

EV

p38

GAPDH

2.5

2.0

1.5

IC50 (nM)

1.0

0.5

0.0

Sh

MAPK

14

Sh

MAPK

14bis

Sh

MAPK

11

Sh

MAPK

12

Sh

MAPK

13

Sh

LUC

HCT116-TP53KO

HCT116-TP53KO

HCT116-TP53KO

NT

SN38

pp38

MAPK11 CA

MAPK12 CA

MAPK14 CA

MAPK13CA

GAPDH

MAPK11 CA

MAPK12 CA

MAPK14 CA

MAPK13CA

HA

p ATF2

tubulin

HCT116-TP53KO

3.5

3.0

2.5

2.0

IC50 (nM)

1.5

1.0

0.5

0.0

MAPK

14

CA

MAPK

11

CA

MAPK

12

CA

MAPK

13

CA

EV

FIGURE 1

A

B

Kinase Assay

C

D

E

F


HCT116-TP53KO EV

HCT116-TP53KO-MAPK14CA

120

100

80

Doubling Time (hours)

60

40

20

0

EV

MAPK14-CA

HCT116-TP53KO

120

HCT116-TP53KO-EV

20

100

HCT116-TP53KO-MAPK14CA

HCT116-TP53 KO

80

15

60

10

% APOPTOSIS

% living cells

40

20

5

0

D7

D14

D21

D28

0

MAPK14

CA

EV

HCT116-TP53KO-EV

HCT116-TP53KO-MAPK14CA

5

4

3

Blue cell number/field

2

1

0

HCT116-

HCT116-

TP53KO-

MAPK14CA

TP53KO

-EV

FIGURE 2

A

B

4

3

Cell number (106)

2

1

0

0

2

4

6

8

10

12

Days

C

D

E


HCT116-TP53KO-EV

HCT116-TP53KO-MAPK14CA

S

37.1%

S

49.4%

G0

G1

28.8%

G0

G1

36.8%

G2/M

19.3%

G2/M

18.4%

% of BrdU stained cells

120

HCT116-TP53KO

1.2

HCT116-TP53KO-EV

100

MAPK14CA

1.0

80

D7

D14

D21

D28

EV

0.8

60

p21

p21 Arbitrary RNA units

0.6

EV

D7

D14

D21

D28

HCT116-TP53KO-MAPK14CA

40

HA

0.4

20

HCT116-TP53KO-Shp21-MAPK14CA

0.2

90

Actin

0

0.0

80

0

5

10

15

20

25

30

70

MAPK14CA

60

HCT116-TP53KO

HCT116-TP53KO-MAPK14CA

50

40

HCT116-TP53KO-ShLuc-

MAPK14CA

shLUC

shp21

30

p21

20

% of BrdU stained cells

10

GAPDH

0

0

5

10

15

20

25

30

HCT116-TP53KO

-ShLuc-MAPK14CA

HCT116-TP53KO

-Shp21-MAPK14CA

S

46.1%

S

25.8%

G0

G1

20.9%

G0

G1

45.6%

G2/M

23.16%

G2/M

28.9%

FIGURE 3

B

A

D

C

E

G

F


HCT116-TP53KO

MAPK14CA

HCT116-TP53KO

D7

D14

D21

D28

EV

EV

MAPK14CA

LC3

Actin

H1299

MAPK14CA

GFP-LC3 labelling

EV

D7 D14 D21

LC3

Tubulin

HCT116-TP53KO

EV

MAPK14CA

500 nm

500 nm

EV

MAPK14CA

HCT116-TP53 KO

6

5

4

Autophagosome

number/cell

3

2

1

0

EV

MAPK14CA

HCT116-TP53 KO

HCT116-TP53KO-MAPK14CA

shLUC

shp21

GFP-LC3 spots/cell

LC3

MAPK14

CA

EV

GAPDH

HCT116-TP53KO

FIGURE 4

B

A

C

D

E

10-

8 -

6 -

4 -

2 -

0 -


HCT116-TP53KO (J14)

HCT116-TP53KO

EV

MAPK14CA

MAPK14CA

NT

2H

4H

8H

NT

2H

4H

8H

EV

D7

D14

D21

D28

p62

p62

GAPDH

2

160

Actin

1

120

p62 level

(arbitrary unit)

80

0

100

EV

80

D7

D14

D21

D28

40

Cell viability (%)

60

p62 level

(arbitrary unit)

0

40

nt

2h

4h

8h

nt

2h

4h

8h

EV

MAPK14CA

20

0

HCT116-TP53KO

MAPK14CA

siCT

siCT

siAtg 5

siAtg 7

siAtg 5

siAtg 7

HCT116-TP53KO

HCT116-TP53KO

3

EV MAPK14CA D14

2.5

HCT116-TP53KO

EV

MAPK14CA D14

2

siRNA

LC3-II level

(arbitrary unit)

HCT116-TP53KO

1.5

LC3

Actin

siCT D2 D4

1

0.5

Atg5

0

NT 3MA Baf NT Baf 3MA

NT

3MA

Baf

NT

3MA

Baf

Atg7

EV MAPK14CA D14

Actin

HCT116-TP53KO

100

80

Cell viability (%)

60

40

20

0

EV MAPK14CA D14

HCT116-TP53KO

FIGURE 5

B

A

C

E

D

NT 3MA Baf NT 3MA Baf


LC3 labelling

HCT116

TP53KO

SW480

Cytotoxic Assay

LC3

4

3

2

1

0

7

NT

SN

NT

SN

6

Actin

NT

LC3

5

SN38 IC50 (nM)

Actin

4

LC3 spots/cell

siCT siAtg5 siAtg7

3

2

1

0

HCT116-TP53KO

NT

SN38

SN38

HCT116-TP53KO

HCT116-TP53 KO

Cytotoxic Assay

Clonogenic Assay

1.6

HCT116-TP53KO

1.2

SN+

Baf

SN+

3MA

SN38 IC50 (nM)

0.8

SN38

8

0.4

LC3

6

0

siCT

siATG5

siATG7

4

SN38 IC50 (nM)

GAPDH

HCT116-TP53KO

2

0

siCT siAtg5 siAtg7

SW480

120

100

80

% living cells

60

40

20

0

NT

SN38

SN38+3MA

HCT116-TP53KO

FIGURE 6

B

A

D

C

E

F

G


HCT116-TP53KO-shMAPK14

HCT116-TP53KO-shLUC

NT SN SN+3MA NT SN SN+3MA

LC3

MAPK14

Actin

B

HCT116-TP53KO

siCT

siATG5

NT

SN38

siATG7

shMAPK

12

shMAPK

13

shMAPK

12

shMAPK

13

shLUC

shMAPK

11

shLUC

shMAPK

11

LC3

GAPDH

3

SN38

SN38+3MA

2

SN38 IC50 (nM)

1

0

HCT116-TP53KO

ShLuc

HCT116-TP53KO

ShMAPK14

3

2

SN38 IC50 (nM)

1

0

HCT116-TP53KO

ShLuc

HCT116-TP53KO

ShMAPK14

FIGURE 7

A

C

D


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