Supplementary Fig. S1 - AACR Journals...2013/05/29 · Days post puro Days post puro A...
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A GEMM Kras +/LSL-G12D Trp53 L/L Kras +/LSL-G12D Trp53 L/L Lkb1 L/L Supplementary Fig. S1 Induce lung tumor A lobe of lung Kras /LSL G12D Trp53 L/L Lkb1 L/L Take nodule A tumor nodule Kras +/G12D Trp53 del/del Lkb1 del/del (Lkb1 null) Homogenize and culture Cell line Kras +/G12D Trp53 del/del (Lkb1-wt) Kras +/G12D Trp53 del/del Lkb1 del/del (Lkb1-null) B H 2 O Tail t5 t4 t2 857 855 634 Cell lines derived from lung tumor nodules Genotype Primer Sequence 5'-3' Kras + mTRR ttctgaccaccaccaacttcaat mTRWTF ctaagccggcactccttacaag Kras + Kras LSL-G12D Trp53 L/L Trp53 del/del mTRWTF ctaagccggcactccttacaag Kras LSL-G12D 5' Kozak 3' LoxP ctagccaccatggcttgagt 3' Flank 3' LoxP tccgaattcagtgactacagatg Trp53 L/L p53-intl-R agcacataggaggcagagac p53-intl-F cacaaaaacaggttaaaccca Trp53 del/del YL9 cacaaaaacaggttaacccag YL10 gaagacagaaaaggggaggg Lkb1 L/L PCR-S5 tctaacaatgcgctcatcgtcatcctcggc LKB 39 gagatgggtaccaggagttggggc Lkb1 L/L Lkb1 +/+ & Lkb1 del/del LKB 39 gagatgggtaccaggagttggggc Lkb1 +/+ & Lkb1 del/del LKB 36 gggcttccacctggtgccagcctgt LKB 37 gatggaggacctcttggccggctca LKB 39 gagatgggtaccaggagttggggc C 634 855 857 t2 t4 t5 LKB1 β-actin
Transcript of Supplementary Fig. S1 - AACR Journals...2013/05/29 · Days post puro Days post puro A...
A
GEMMKras+/LSL-G12DTrp53L/L
Kras+/LSL-G12DTrp53L/LLkb1L/L
Supplementary Fig. S1
Induce lung tumor
A lobe of lung
Kras /LSL G12DTrp53L/LLkb1L/L
Take nodule
A tumor nodule
Kras+/G12DTrp53del/delLkb1del/del (Lkb1 null)
Homogenize and culture
Cell lineKras+/G12DTrp53del/del (Lkb1-wt)
Kras+/G12DTrp53del/delLkb1del/del (Lkb1-null)
B
H2O
Tai
l
t5t4t2857
855
634
Cell lines derived fromlung tumor nodules
Genotype Primer Sequence 5'-3'
Kras+ mTRR ttctgaccaccaccaacttcaat
mTRWTF ctaagccggcactccttacaag
Kras+
KrasLSL-G12D
Trp53L/L
Trp53del/del
mTRWTF ctaagccggcactccttacaag
KrasLSL-G12D 5' Kozak 3' LoxP ctagccaccatggcttgagt
3' Flank 3' LoxP tccgaattcagtgactacagatg
Trp53L/L p53-intl-R agcacataggaggcagagac
p53-intl-F cacaaaaacaggttaaaccca
Trp53del/del YL9 cacaaaaacaggttaacccag
YL10 gaagacagaaaaggggaggg
Lkb1L/L PCR-S5 tctaacaatgcgctcatcgtcatcctcggc
LKB 39 gagatgggtaccaggagttggggcLkb1L/L
Lkb1+/+ & Lkb1del/del
LKB 39 gagatgggtaccaggagttggggc
Lkb1+/+ &
Lkb1del/del
LKB 36 gggcttccacctggtgccagcctgt
LKB 37 gatggaggacctcttggccggctca
LKB 39 gagatgggtaccaggagttggggc
C
634
855
857
t2 t4 t5
LKB1
β-actin
Supplementary Fig. S1
A549 NIH3T3D
t2 t5t4
10 μm
634 855 857
• Anti-Pancytokeratin (Red) to stain epithelial cells
• DAPI (Blue) to stain nucleus
• All images are merged signals
E
gro
wth
nt r
eado
ut)
1000000
1250000
1500000
634
855 15
20
25
tim
e (h
ou
r)
Rel
ativ
e g
(Lum
ines
cen
Hour
0
250000
500000
750000
0 10 20 30 40 50 60
857
t2
t4
t50
5
10
15
634 855 857 t2 t4 t5
18.4
20.2
18.3
17.2
21.1
20
Cel
l do
ub
ling
t
F
0.6
0.8
1
1.2
200000
300000
400000
AT
P l
evel
sce
nt r
eado
ut)
AT
P l
evel
svs
. 63
4)
P<0.005
P<0.005
0
0.2
0.4
634 t40
100000
634 855 857 t2 t4 t5
Rel
ativ
e (lu
min
esc
Rel
ativ
e (F
old
v
Supplementary Fig. S2
100
63.7
77.960
80
100
120
e D
TY
MK
cr
ipts
(%
) A
β-actin
DTYMK20.027.8
51.1
0
20
40
Rel
ativ
tran
sc
B
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
shD
tym
k-1
shD
tym
k-3
634 855 857 t2 t4 t5
DTYMK
β-actin
Supplementary Fig. S3
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
+ d
TT
P
shD
tym
k-1
+ d
TT
P
shD
tym
k-3
+ d
TT
P
shG
FP
shD
tym
k-1
shD
tym
k-3
shG
FP
+ d
TT
P
shD
tym
k-1
+ d
TT
P
shD
tym
k-3
+ d
TT
PA
DTYMK
β-actin
s s s s s s s s s s s s
634 t4634 t4
25000TP
m
)
B
0
5000
10000
15000
20000
3H-dT
3H-dTTP
3H-dTTP/non cell control
corp
ora
tio
n o
f 3H
-dT
Tr
3H
-dT
) in
to D
NA
(cp
(n=
6)
0
634 t4
In (o
Supplementary Fig. S4
CHIR124 ( 20 nM, 3h)+ + ++ + +AZD7762 (100 nM, 3h)+ + ++ + +DMSO+ + ++ + +
γH2AX
A
634 855 857 t2 t4 t5
β-actin
B
+ + + + AZD7762 (150 nM, 3h)+ + + + DMSO
Calu-1H358 H2122 A549
+ + + + CHIR124 (150 nM, 3h)
β-actin
γH2AX
Supplementary Fig. S5
AP<0.00001
X
5
10
15
20
25
30
35
er o
f ce
lls w
ith
γH
2AX
mic
rosc
op
ic f
ield
0
5
Nu
mb
ep
er
KN4147 mouse lung tumor (Kras/p53)
KNC125_306 mouse lung tumor (Kras/p53/Lkb1)
C
LKB1
B
Ph
osp
ho
-CH
EK
1 P
rote
in L
evel
β-actin
γH2AX
t-CHEK1
DTYMK
LKB1: wt MutantLkb1-wt Lkb1-null
β actin
Supplementary Fig. S6
TY
MK
-D3
TY
MK
-D8
TY
MK
-D10
TY
MK
-1
TY
MK
-3
FP
A
40
60
80
100
e D
TY
MK
cr
ipts
(%
)
DTYMK
β-actin
shD
T
shD
T
shD
T
shD
T
shD
T
shG
F
0
20
40
Rel
ativ
tran
scL
evel
B
CH
EK
1 P
rote
in L
Supplementary Fig. S7
Lkb1-wt versus Lkb1-null cells
Less DTYMK (Lkb1-null)Normal DTYMK (Lkb1-wt)
Deplete DTYMK
beginning
Deplete DTYMK moderately
Deplete DTYMKmostly
Stop dTTP and DNA synthesis
Thymine-less cell death
High ratio of dUTP/dTTP
Uracil in DNA and DNA-DSBs
Higher ratio of dUTP/dTTP
More Uracil in DNA and DNA-DSBs
Checkpointactivation
Checkpointactivation
Base excision repairand DSB repair
Increased base excision repair
and DSB repair
Supplementary Fig. S8
AA
mk
tran
scri
pts
VS
. 63
4)
P=0.01
80
100
120
140R
elat
ive
Dty
m(%
) (V
0
20
40
60
BP<0.01
120
rip
ts
20
40
60
80
100
ive
Ch
ek1
tran
scr
(%)
(VS
. 63
4)
0
Rel
at
Supplementary Fig. S9
120A
Rel
ativ
e T
K1
tran
scri
pts
(%
)
0
20
40
60
80
100
120
B1200000
6341500000
t2
0
300000
600000
900000
1 2 3 4
634
0
300000
600000
900000
1200000
1 2 3 4
t2
shGFP
shTK1-3
shTK1-4
Cel
l gro
wth
300000
600000
900000
1200000855
300000
600000
900000
1200000
1500000t4
Cel
l gro
wth
0
1 2 3 40
1 2 3 4Days post puro Days post puro
A
Supplementary Fig. S10
DTYMK expressionin Beer dataset
og
2 m
edia
n-c
ente
red
inte
nsi
ty
P = 4.9·10-7
FC = 2.1
in Beer dataset
BCAN/DF dataset
lL
o
Normal lung(n=10)
Lung adenocarcinoma(n=86)
Log rank P = 0.01Cox P = 0.004
Month
Su
rviv
al
High DTYMK (n=20)
Low DTYMK (n=62)
Log rank P = 0.02Cox P = 0.03
Beer dataset
High DTYMK (n=54)
Low DTYMK (n=30)
Su
rviv
al
Month
NCCRI dataset
Low DTYMK (n=153)
viva
l
Log rank P = 0.0001Cox P = 0.0005
Day
High DTYMK (n=51)
Su
rv
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