Figure 1

A

-SN50 -C8-S -A2 -G7

B

C

HCT116-s HCT116-SN6

NT SN38 NT SN38

HCT116-s

pp38

tubulin

NT 0.1µM 1µM 5µM 10µM

pp38

tubulinHCT116-SN6

[SN38] 24H

pp38

NT 1h 4h 7h 16h 24h 48h

tubulin

pp38

tubulin

HCT116-s

HCT116-SN6

SN38 1µM

HCT116-SN6

Figure 2

A B C

0

1

2

3

Luc

HCT116-s

p38 Sh RNAIC

50 (

nM)

0

1

2

3

EV

HCT116-s

p38 CA isoforms

IC50

(nM

)

F

IC50

(nM

)

HCT116-SN6

0

5

10

15

20

25

Luc

p38 Sh RNA

G

0

5

10

15

20

25

EV

p38 CA isoforms

HCT116-SN6

IC50

(nM

)

p38δ

p38α

p38

p38

ShLuc

Shp38

EV p38CA

HCT116-s

-Actin

E

p38δ

p38α

p38

p38

ShLuc

Shp38

EV p38CA

HCT116-SN6

-Actin

p< 0.05, p< 0.01, p<0.001

HCT116-s

IC50

(nM

)

0

1

2

3

EV +

p38 CA isoforms

HCT116-SN6

IC50

(nM

)

0

4

8

12

16

EV

p38 CA isoforms

H

D

Figure 3

A

E

01234567

SN38 SN38+SB

SW480

IC50

(nM

)

IC50

(nM

)

0

2

4

6

8

10

12

SN38 SN38+SB

HT29

B

C

0

1

2

3

4

IC50

(µ

M)

5-FU 5-FU+SB

HCT116-s

0

200

400

600

IC50

(n

M)

Oxali Oxali+SB

HCT116-s

pp38

-Actin

p38

NT 5-FU Oxali

HCT116-s HCT116-SN6

NT SN38 SN38 + SB

NT SN38 SN38 + SB

pATF2

F

p< 0.05, p< 0.01, p<0.001

IC50

(nM

)

0

2

4

6

8

10

12

EV p38

SW480

IC50

(nM

)

p38 CA isoforms

HT29

0

2

46

810

1214

EV p38

p38 CA isoforms

D

HCT116-SN6

SN

38 IC

50 (

nm)

SN38 SB SN38+SB

SB

IC50

(µ

m)

0

2

4

6

8

10

0

5

10

15

20

25

HCT116-s

SN38 SB SN38+SB

SN

38 IC

50 (

nM)

0,0

0,5

1,0

1,5

2,0

0

5

10

15

20

SB

IC50

(µ

M)

Figure 4

0

200

400

600T

umor

siz

e (m

m3)

1 5 9 12 15 18Days

HCT116-SN6HCT116-SN6 + IrinotecanHCT116-sHCT116-s + Irinotecan

Irinotecan 40 mg/ml IP

0

200

400

600

800

Tum

or s

ize

(mm

3)

0 5 10 15 20Days

NTIrinotecanSB202190I+SB202190

A B

Irinotecan 40 mg/ml IP

SB202190

p< 0.05, p< 0.01, p<0.001

C

% m

ice

with

tum

or s

ize

doub

led

0

25

50

75

100

0 5 10 15 20 25 30Days

NTIrinotecanSB202190I+SB202190

100

75

50

25

0

0 5 10 15 20 25 30Days

Figure 5

A B

C

50

100

150

200

250

300QS

nucle

ar

NR R

Response

nuclear QS

ResponderNon

ResponderTotal

<147 8 5 13

>147 1 7 8

Total 9 12 21

1

2

3

4

5

6

SW

48-s

SW

48-S

N1

SW

48-S

N2

SW

48-S

N3

SW

48-S

N4

pp38

tubulin

  IC50 (nM)Resistance

Factor

SW48-s 6,1± 0,8 1

SW48-SN1 377,5 ± 37 62

SW48-SN2 262,0 ± 24 43

SW48-SN3 121,0 ± 17 20

SW48-SN4 279,3 ± 53 46

A B

Supplemental Figure 1: p38 is activated by phosphorylation in SN38-resistant SW48 cells. A: Drug sensitivity of the SW48 clones: IC50 values were determined using the SRB assay; the resistance factor was determined by dividing the IC50 value of each resistant clone by that of the sensitive clone SW48-s. Data represent the mean ±SD of at least 3 independent experiments. B: Western blot analysis of p38 phosphorylation in SW48-s cells and in the SN38 resistant clones SW48-SN1, SW48-SN2, SW48-SN3, SW48-SN4.

A

C

Clone 1 Clone 2

MKK6 CA EV

SW480

FLAG

pATF2

WB

KA

0

1

2

3

4

5

6

7

8

EV MKK6 CAClone 1

MKK6 CAClone 2

IC50

(nM

)

SW480

IC50

(nM

)

HCT116

0

1

2

3

4

EV MKK6 CA

FLAG

MKK6 CAEV

WB

HCT116-s

pp38WB

tubulin

p38WB

WB

Supplemental Figure 2: Analysis of the involvement of MKK6 CA in SN38 resistance. A: Western blot analysis of HCT116 cells that express constitutively active (CA) MKK6 (or EV, as a control). MKK6 overexpression was detected with anti-FLAG antibody. p38 expression and activation were detected with anti-p38 and anti-phospho-p38 antibodies respectively. Equal loading is shown by tubulin expression. B: SRB assay on HCT116 expressing MKK6 CA or EV as control, and treated with SN38. C: Western blot analysis of 2 clones of SW480 cells that stably express constitutively active (CA) MKK6 (or EV, as a control). MKK6 overexpression was detected with anti-FLAG antibody. p38 activation was detected using a kinase assay to test p38a activity in the 2 clones stably expressing MKK6 CA or Empty Vector as a control. D: SRB assay on SW480 expressing MKK6 CA or EV as control, and treated with SN38.

B

D

A C

B

-actin

shLu

c

Shp

38α

pMS

CV

pMS

CV

p38β

shLu

c

Shp

38α

pMS

CV

pMS

CV

p38β

Topo I

HCT116-s HCT116-SN6

1.3 1.2 1.6 2.3 1.3 1.5 1.7 2.5

Sh Luc Shp38

HCT116-SN6HCT116-s

Sh Luc Shp38

NT

Sn381µM 0

20

40

60

80

100

120

140

ShLuc Shp38

HCT116-SN6HCT116-s

ShLuc Shp38IC

E a

rbitr

ary

units

SN38 treated samples

HCT116-SN6

pMSCV p38

NT

Sn381µM

HCT116-SN6

pMSCV p38 0

20

40

60

80

100

120

140

ICE

arb

itrar

y un

its

SN38 treated samples

Supplemental Figure 3: Analysis of Topoisomerase I expression and activity. A: Western blot analysis of Topoisomerase I (TopoI) in HCT116-s-ShLuc, HCT116-s-Shp38a, HCT116-s-pMSCV and HCT116-s-CAp38b cells and in HCT116-SN6-ShLuc, HCT116-SN6-Shp38a, HCT116-SN6-pMSCV and HCT116-SN6-CAp38b cells. Equal loading is shown by b-Actin expression . Numbers underneath the b-actin panel are the quantification data for total TopoI level obtained from western blot analysis after normalization to β-actin. B: Quantification of SN38-induced TopoI-DNA complexes using the ICE bioassay and nuclear extracts from HCT-116-s-ShLuc and -Shp38a cells and HCT116-SN6-ShLuc and -Shp38a cells. The relative intensity of the immune complexes in SN38-treated cells was normalized to that of untreated cells. C: Quantification of SN38-induced TopoI-DNA complexes using the ICE assay and nuclear extracts from HCT116-SN6-pMSCV and -CAp38b cells. The relative intensity of the immune complexes in SN38-treated cells was normalized to that of untreated cells.

A B

1 2 3 4 5 6 7 8

pMSCV p38 beta

-actin

p38 D

Days

Tum

or s

ize

(m

m3 )

0

100

200

300

400

500

600

0 5 10 15 20 25

HCT116-SN6 pMSCVHCT116-SN6 p38

Irinotecan 40 mg/ml IP

Days

Tum

or s

ize

(m

m3 )

0

100

200

300

400

500

600

0 5 10 15 20 25

HCT116-SN6 Sh LucHCT116-SN6 Sh p38

Irinotecan 40 mg/ml IP

C

0

100

200

300

400

500

600

0 5 10 15 20 25

HCT116-SN6 pMSCVHCT116-SN6 p38 CAHCT116-SN6 Sh LucHCT116-SN6 Sh p38

Days

Tum

or s

ize

(mm

3)

1 2 3 4 5 6 7 8

Sh Luc Sh p38

-actin

p38 E

HCT116-SN6

0102030405060

Sh Luc Sh p38 alpha

p38 /-actin

Arb

itrar

y un

its

Supplemental Figure 4: The differential expression of the four p38 isoforms influences the response to irinotecan. A: Tumor growth kinetics in mice xenografted with HCT116-SN6- pMSCV or HCT116-SN6-p38, HCT116-SN6-ShLuc or HCT116-SN6-Shp38 cells before irinotecan treatment. B: Tumor growth kinetics in mice xenografted with HCT116-SN6-ShLuc or HCT116-SN6-Shp38 cells and treated with irinotecan. C: Tumor growth kinetics in mice xenografted with HCT116-SN6-pMSCV or HCT116-SN6-p38 cells and treated with irinotecan. D: Western blot analysis of p38 expression in HCT116-SN6-pMSCV and HCT116-SN6-CA38 xenografts. Equal loading is shown using -Actin. E: Western blot analysis of p38expression in HCT116-SN6-ShLuc or HCT116-SN6-Shp38 xenografts. Equal loading is shown using -Actin. Histogram shows the quantification data for p38 level obtained from western blot analysis after normalization to β-actin.