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.