Figure 1
[SN38] 24H
SN38 1µM
NT
A
1h
4h
7h
16h
24h
NT
48h
HCT116-s
tubulin
tubulin
pp38
HCT116-SN6
HCT116-SN6
tubulin
B
5µM 10µM
pp38
pp38
HCT116-s
0.1µM 1µM
pp38
tubulin
HCT116-s
HCT116-SN6
HCT116-SN6
NT
C
-S
SN38
-A2
NT
-SN50
SN38
-C8
-G7
Figure 2
HCT116-s
A
B
3
C
HCT116-s
3
D
HCT116-s
HCT116-s
3
p38 g
2
1
IC50 (nM)
p38 b
IC50 (nM)
IC50 (nM)
p38α
2
1
p38δ
b-Actin
Luc
p38
CA
b
g
d
25
p38α
IC50 (nM)
p38 g
G
HCT116-SN6
p38 CA isoforms
H HCT116-SN6
16
20
15
10
15
10
5
b-Actin
0
0
Luc
p38
CA
EV a + b
d
25
5
EV
g
HCT116-SN6
p38δ
Sh Sh
Luc p38
b
p38 CA isoforms
20
p38 b
a
EV
p38 Sh RNA
F
HCT116-SN6
a
IC50 (nM)
E
EV
a
b
g
p38 Sh RNA
d
IC50 (nM)
Sh Sh
Luc p38
1
0
0
0
2
12
8
4
0
EV
a
b
g
p38 CA isoforms
 p< 0.05,  p< 0.01, p<0.001
d
EV a + b
p38 CA isoforms
Figure 3
0,5
5
0
0,0

15
4
10
2
5
0
B
HCT116-s
pATF2
NT
SN38
0
12
SW480
SN38
+ SB
NT
HT29
10
12
10
8
6
4
SN38
SN38+SB
8
6
4
2
0
0
SN38+SB
EV
p38 a+b
14
12
10
8
6
4
2
0
p38 CA isoforms
pp38
p38
b-Actin
SN38
+ SB
Oxali
4
F
HCT116-s
3
2
1
0
p38 a+b
600
HCT116-s
400
200
0
5-FU
 p< 0.05,  p< 0.01, p<0.001
EV
p38 CA isoforms
IC50 (nM)
E
5-FU
IC50 (µM)
NT
SN38
HT29
SW480
D
2
SN38
HCT116-SN6
SN38 SB SN38
+SB
IC50 (nM)
IC50 (nM)
C

6
SN38 SB SN38
+SB
7
6
5
4
3
2
1
0
20
IC50 (nM)
 10
1,0
8
25
IC50 (nM)
15
SN38 IC50 (nm)
1,5
SB IC50 (µM)

HCT116-SN6
SB IC50 (µm)
10
HCT116-s 20
2,0
SN38 IC50 (nM)
A
5-FU+SB
Oxali
Oxali+SB
Figure 4
B
800
400
200
400
200
0
1
5
9
12
15
NT
Irinotecan
SB202190
I+SB202190
600
Tumor size (mm3)
HCT116-SN6
HCT116-SN6 + Irinotecan
HCT116-s
HCT116-s + Irinotecan
600
0
18
0
Days
5
10
15
20
Days
SB202190
Irinotecan 40 mg/ml IP
Irinotecan 40 mg/ml IP
C
% mice with tumor size doubled
A
100
0
NT
Irinotecan
SB202190
I+SB202190
75
25
50
50
25
75
0
100 0
0
5
5
10
10
15
Days
15
Days
20
20
25
25
30
30
 p< 0.05,  p< 0.01, p<0.001
Figure 5
300
A
B
1
4
2
5
3
6
QS nuclear
250
200
150
100
50
NR
R
C
Response
nuclear
QS
Responder
Non
Responder
Total
<147
8
5
13
>147
1
7
8
Total
9
12
21
SW48-SN1
377,5 ± 37
62
SW48-SN2
262,0 ± 24
43
SW48-SN3
121,0 ± 17
20
SW48-SN4
279,3 ± 53
46
SW48-SN4
1
SW48-SN3
6,1± 0,8
SW48-SN2
SW48-s
B
SW48-SN1
IC50 (nM)
Resistance
Factor
SW48-s
A
pp38
tubulin
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, SW48SN2, SW48-SN3, SW48-SN4.
HCT116-s
B
MKK6 CA
EV
WB
4
FLAG
HCT116
IC50 (nM)
A
pp38
WB
WB
p38
3
2
1
0
EV
WB
tubulin
D
SW480
MKK6 CA
Clone 1
KA
EV
Clone 2
FLAG
pATF2
IC50 (nM)
C
WB
MKK6 CA
8
7
6
5
4
3
2
1
0
SW480
EV
MKK6 CA
Clone 1
MKK6 CA
Clone 2
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.
C
pMSCVp38β
HCT116-SN6
NT
Topo I
Sn38
1µM
b-actin
1.3
1.2 1.6
2.3
1.3
1.5
SN38 treated samples
pMSCV p38 b
ICE arbitrary units
pMSCV
Shp38α
shLuc
HCT116-SN6
pMSCVp38β
pMSCV
shLuc
Shp38α
HCT116-s
A
1.7 2.5
140
120
100
80
60
40
20
0
pMSCV p38 b
HCT116-SN6
HCT116-s
B
HCT116-SN6
Sh Luc Shp38 a Sh Luc Shp38 a
Sn38
1µM
ICE arbitrary units
NT
140
SN38 treated samples
120
100
80
60
40
20
0
ShLuc Shp38a ShLuc Shp38a
HCT116-s
HCT116-SN6
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, HCT116SN6-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-sShLuc and -Shp38a cells and HCT116-SN6-ShLuc and -Shp38a cells. The relative intensity of the immune complexes in SN38treated 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
Tumor size (mm 3)
500
400
300
200
100
600
HCT116-SN6 Sh Luc
HCT116-SN6 Sh p38 a
500
400
300
200
5
10 Days 15
20
400
300
200
100
100
0
0
0
25
HCT116-SN6 pMSCV
HCT116-SN6 p38 b
500
0
5
10
15
20
0
25
5
10
15
Days
E
p38 b
b-actin
1
2
3
pMSCV
4
5
6
7
8
p38 beta
p38 a
b-actin
1
25
Irinotecan 40 mg/ml IP
Irinotecan 40 mg/ml IP
D
20
Days
Arbitrary units
Tumor size (mm 3)
HCT116-SN6 pMSCV
HCT116-SN6 p38 b CA
HCT116-SN6 Sh Luc
HCT116-SN6 Sh p38 a
Tumor size (mm3)
600
600
0
C
2
3
Sh Luc
4
5
6
Sh p38 a
7
8
60
50
40
30
20
10
0
p38 a /b-actin
Sh Luc
Sh p38 alpha
HCT116-SN6
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-p38b, HCT116-SN6-ShLuc or HCT116-SN6Shp38a cells before irinotecan treatment. B: Tumor growth kinetics in mice xenografted with HCT116-SN6-ShLuc or HCT116SN6-Shp38a cells and treated with irinotecan. C: Tumor growth kinetics in mice xenografted with HCT116-SN6-pMSCV or
HCT116-SN6-p38b cells and treated with irinotecan. D: Western blot analysis of p38b expression in HCT116-SN6-pMSCV
and HCT116-SN6-CA38b xenografts. Equal loading is shown using b-Actin. E: Western blot analysis of p38a expression in
HCT116-SN6-ShLuc or HCT116-SN6-Shp38a xenografts. Equal loading is shown using b-Actin. Histogram shows the
quantification data for p38a level obtained from western blot analysis after normalization to β-actin.