Figure S1 A MRC5 - + - + - + - + - + - + - + Control Chk1 TdR(h): 30 1 6 16 24 30 48 γH2AX Cleaved...
-
Upload
adela-horn -
Category
Documents
-
view
219 -
download
1
Transcript of Figure S1 A MRC5 - + - + - + - + - + - + - + Control Chk1 TdR(h): 30 1 6 16 24 30 48 γH2AX Cleaved...
Figure S1
A MRC5
- + - + - + - + - + - + - +
Control Chk1
TdR(h): 30 1 6 16 24 30 48
γH2AX
Cleaved casp3
RPA34
Chk1
β-actin
siRNA:
B SW480
- + - + - + - + - + - +
Control Chk1
TdR(h): 30 1 6 24 30 48
γH2AX
Cleaved casp3
RPA34
Chk1
β-actin
siRNA:
TdR(h): 30 1 6 16 24 30
γH2AX
Cleaved casp3
RPA34
Chk1
β-actin
- ---- -+ + + + + +
Chk1 inhibitor (Gö6976) 0.1 M
10Gy
C
Figure S1. H2AX formation is enhanced in Chk1-depleted or Chk1 inhibited cells during replication stress. Western blot analysis of γH2AX, RPA 34 and cleaved caspase 3 in extracts obtained from MRC5VA (A) or SW480 (B) cells transfected with control or Chk1 siRNAs and treated or not treated with 2 mM thymidine for the indicated times. (C) Western blot analysis of extracts from HCT116 cells treated with the Chk1 inhibitor. Levels of H2AX and RPA34 in extracts of cells exposed to 10 Gy IR are also presented. The levels of Chk1 in the all the cells are presented and -actin levels are presented as loading controls.
HCT116
Figure S2
A
B
γH2AX
RPA34
Merge
Control HCT116 cells
γH2AX
RPA34
Merge
Min post IR - 15 30 60 90 120
Chk1 depleted HCT116
Figure S2. Induction of γH2AX formation and foci in Chk1 depleted cells exposed to IR. (A) Western blot analysis of H2AX phosphorylation in HCT116 cells treated with control or Chk1 siRNAs for 24 hours before IR. Cells were harvested at the indicated times post IR. Chk1 depletion had little effect on the transient formation of H2AX following IR. (B & C) Representative images of IR-induced γH2AX foci formation at the indicated times in control (B) or Chk1 (C) siRNA transfected HCT116 cells. Nucleus is co stained for RPA34. Formation of H2AX foci occurs at 15 to 90 minutes post IR exposure and is not affected by Chk1 depletion, while RPA foci are not detected. Thus the nuclear organization of H2AX and RPA34 are distinctly different in cells exposed to IR relative to those exposed to replication stress.
Chk
1
Chk
1
Chk
1
Chk
1
Chk
1
Con
trol
Con
trol
Con
trol
Con
trol
Time(min) after IR (10Gy): - - 30 30 60 60 90 90 120 120
γ-H2AX
RPA34
Chk1
β-actin
siRNA:
Con
trol
Min post IR - 15 30 60 90 120
C
Figure S3
Time(h) in thymidine
05
1015
2025
3035
4045
50
0 10 20 30 40 50 60
% c
ells
cle
ave
d c
asp
-3
Chk1-depleted -TdRChk1-depleted +TdRControl-depleted -TdRControl-depleted +TdR
A
05
10
1520253035
404550
0 10 20 30 40 50 60
% T
UN
EL
+ c
ells
Time(h) in thymidine
B
Figure 3. Induction of apoptosis in Chk1 depleted HCT116 cells following thymidine treatment. Cultures of HCT116 cells transfected with control or Chk1 siRNAs were treated with 2mM thymidine for the indicated times or left untreated as controls. Cells were then harvested and the level of apoptotic cells was determined by flow cytometry measuring the percentage of cells with active caspase-3 (A) or TUNEL+ (B). Results represent the
means of three independent experiments + standard deviations.
HCT116 Chk1-depleted cells
DNA γH2AX merge
II
II
II
II
I
II
II
IIII
III
III
I
I
I (1-10 foci)
II (>10 foci)
III (diffused
pan nuclear staining)
TdR (h):
6h
6h
6h
16h
16h
16h
Figure S4
Figure S4. Representative images of H2AX stained HCT116 cells that were depleted of Chk1 and treated with 2mM thymidine for the indicated times before analysis. H2AX forms distinct foci at early times (6h) that becomes more diffuse at longer exposures (>16h). Cells with pan-nuclear staining (III) become more frequent at these later times.
Figure S5
A
B
C
-H2AX
RPA34
β-actin
Chk1
Cdc45
ControlsiRNA:
TdR (24h): - + - + - - - -
Cdc45 Chk1Chk1
Cdc45
SW480
Cdc45
Chk1
- 2H AX
34RPA
116HCT
-actin
6976 (0.1Gö ):M
- + - + - + - +
- - - - - - - -
- + - +
+ + + +
TdR (24 ):h
ControlsiRNA: 45Cdc 1Chk1Chk45Cdc Control 45Cdc
HCT 116
-actin
- 2H AX
1Chk
45Cdc
34RPA
1Chk1 +Chk
45 (2)Cdc
TdR 24h -
Control 45 (2)Cdc
- - -+ + + +siRNA:
Figure 4. Cdc45 suppresses the enhanced induction of H2AX phosphorylation during DNA replication stress in the absence of Chk1. Western blot analysis of γH2AX and RPA34 in extracts obtained from the indicated cells transfected with control, Cdc45, or Chk1 siRNAs or treated with the Chk1 inhibitor and exposed to 2 mM thymidine for 24h. The levels of Chk1 and Cdc45 proteins in the cells also are presented, while -actin levels are presented as loading controls. (A) HCT116 cells treated a second Cdc45 siRNA. (B) HCT116 cells treated with the Chk1 inhibitor. (C) SW480 cells treated with Cdc45 and/or Chk1 siRNAs.
Figure S6
A
control
untreated
Cdc45 Chk1/Cdc45Chk1
24hTdR
γH2A
X controlChk1
Cdc45Chk1/Cdc45
DNA content
controlChk1
Cdc45Chk1/Cdc45
DNA content
Gated R3 region cells
DNA content
6.5% 5.2% 5.6% 10.9%
11.3% 58.3% 9.1% 14.4%
B
Figure S6. Cdc45 depletion suppresses the enhanced formation of γH2AX in Chk1 depleted cells treated with thymidine and the fraction of cells in mid S-phase. (A) Scatter plots present the flow cytometric analysis of γH2AX levels and DNA content (propidium iodide staining) in HCT116 cells transfected with the indicated siRNAs and cultured in thymidine-free (top) or thymidine containing (bottom) media for 24h. Cell cycle profiles (filed histograms below the corresponding γH2AX assay) for all cells analysed in this experiment. (B) Histograms presenting DNA content of cells staining for increased γH2AX (boxed R3 region in A).
siRNA:
32
40
48
64
24
0
Hours in TdR
control Chk1
Release in TdR-free medium (h)
8
16
24
40
Continuous exposure to TdR(h)control Chk1control Chk1
0
24
Hours in TdR
0%
20%
40%
60%
80%
100%
control Chk1 G2/MSG1SubG1
0%
20%
40%
60%
80%
100%
32
40
48
64
Continuous exposure to TdR(h)
Release in TdR-free medium (h)
control Chk1control Chk1
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
8
16
24
40
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
0%
20%
40%
60%
80%
100%
Figure S7
Figure S7. Chk1-depleted cells show a reduced ability to re-enter the cell cycle after release from thymidine treatment. (A) Flow cytometric analysis of DNA content of HCT116 cells transfected with control or Chk1 siRNAs and treated or not treated with 2mM thymidine (TdR) for 24h (top panels). Cells were then either washed and transferred to fresh medium without thymidine (right panels) or cultured in the continued presence of thymidine (left panels) for the indicated times. (B) Bar graphs presenting the cell cycle distributions of HCT116 cells treated as described in panel A. The results are the means of three independent experiments + standard deviations.
A
B
untreated
2.7%
Control siRNA
1.4%
40hTdR
4.6% 31.2%
3.2% 24.1%
DNA content
Act
ive
casp
-3Chk1 siRNA
24hTdR,
24h Release
Figure S8
Figure S8. Activation of caspase 3 in Chk1-depleted cells released from thymidine. Scatter plots present levels of cleaved caspase 3 and DNA content (propidium iodide staining) determined by flow cytometry in control or Chk1 siRNA transfected HCT116 cells, continuously exposed to thymidine (TdR) for 40h or exposed for 24h and then released into thymidine-free medium for 24h. Untreated cells were used as negative controls. The percentages of cells with activated caspase-3 (boxed R4 region) are presented.
Figure S9
A
Chk1 siRNA
0h6h
46h6h, R40h
BControl siRNA Control siRNA
0h6h
46h6h, R40h
0h16h
30h16h, R30h
Chk1 siRNA
0h16h
30h16h, R30h
Figure S9. Chk1-depleted cells recover from a short (6h) exposure to thymidine but only show partial recovery from a 16h arrest. HCT116 cells transfected with control or Chk1 siRNAs were treated with 2mM thymidine (TdR) for 6h (A) or 16h (B) and then released into thymidine-free medium for the indicated times or were left to grow continuously in the inhibitor before being harvested and analysed for DNA content. Control cells thymidine-arrested were able to recycle and fully recover from replication stress after release in a thymidine-free medium. Chk1 depleted cells exposed to thymidine for 6h recovered completely after release but those exposed for 16h showed only a partial recovery. Cells transfected with the control siRNA recovered completely after 6 or 16h exposure.
G1: 5.7
S: 27.1
G2: 4.56
37.4% G1: 14.4
S: 64.1
G2: 10.73
89.3% G1: 34.9
S: 24.2
G2: 27.65
86.7% G1: 10.9
S: 22.9
G2: 2.48
36.2% G1: 10.2
S: 21.4
G2: 11.54
43.2%
SW480 24h thymidine
Control siRNA Chk1 siRNA
h post BrdU pulse
Total cells
G1: 6.2
S: 28.9
G2: 53.44
88.6%
0 4 24 0 4 20
Figure S10
G1: 7.76
S: 5.13
G2: 3.66
16.5% G1: 58.84
S: 29.60
G2: 5.05
93.5%G1: 6.99
S: 73.52
G2: 12.69
93.2% G1: 30.25
S: 22.03
G2: 40.03
92.3% G1: 11.22
S: 3.40
G2: 1.74
16.4% G1: 6.06
S: 6.70
G2: 2.44
15.2%
SW480 24h HU
Control siRNA Chk1 siRNA
h post BrdU pulse
0 4 24 0 4 20
A
B
Figure S10. Chk1 depleted SW480 cells released from thymidine treatment show suppressed re-entry into S-phase. SW480 cells transfected with control or Chk1 siRNAs were exposed to 2mM thymidine (A) or 2mM HU (B) for 24h. The cells were then washed with inhibitor-free medium and transferred to medium containing 10 M BrdU. After 1h these cells were washed with BrdU-free medium and harvested for FACS analysis at the indicated times. Representative scatter plots (upper panels) show BrdU incorporation and DNA content (PI staining) in cells transfected with control or Chk1 siRNAs treated with thymidine (A) or HU (B). Cells incorporating BrdU have been gated (as indicated) and the percentages with G1, S, or G2 DNA content are presented together with the percentages of all cells incorporating BrdU. Cells with a G1 DNA content incorporating BrdU at early times after the pulse most likely represent cells at the G1/S border. Lower panels present DNA content (PI staining) of all the cells in the cultures (gated and ungated).
B
SW480
Control siRNA
Chk1 siRNA
Release (h): - - 24
TdR (h): - 24 24
γ-H2AX γ-H2AX
γ-H2AX
γ-H2AX
γ-H2AX γ-H2AX
% c
ells
with
H
2AX
foci
untreated TdR24h TdR24h
R24h
Control Chk1
siRNA: Control
Chk1Control
Chk1
1-10 foci
>10 foci
pan nuclear
0
10
20
30
40
50
60
C
A
D
Figure S11
TdR(24h): HU(24h) IR(10Gy)
Release(24h)
Chk1
pSer1981 ATM
Control siRNA:
pThr68 Chk2
β-actin
γH2AX
Chk1
RPA34
- - + + - - - + + - - - - - - + + - - - + + + - - - - - - - - - - - - - + - + - - + - +
0
10
20
30
40
50
60
70
80
90
100
TdR(h): 0 0 24 24 24 24 24 24
Release (h): 0 0 0 0 24 24 40 40
Control Chk1 siRNA: Control Control Control Chk1 Chk1 Chk1
H2AX+
H2AX+ with SubG1
% c
ells
Release. (h): 0 0 0 0 24 24 40 40
010
20
304050
60
70
80
90
100
Control Chk1 siRNA: Control Control Control Chk1 Chk1 Chk1
HU(h): 0 0 24 24 24 24 24 24
% c
ells
Figure S11. Persistence of H2AX and other DNA damage response proteins in Chk1-depleted SW480 cells following release from thymidine arrest. (A) Western blot analysis of γH2AX, RPA34, phospho-ATM (pSer1981), and phospho-Chk2 (pThr68) in extracts obtained from SW480 cells transfected with control or Chk1 siRNAs and treated or not treated with 2 mM thymidine (TdR) or 2mM HU for 24h before release into thymidine-free medium for the indicated times. In addition extracts from SW480 cells treated with 10 Gy IR and then cultured for 1h before harvest are included. The levels of Chk1 protein in all the cells also are presented while -actin levels are presented as loading controls. (B) Representative immunofluorescence images of γH2AX nuclear distribution in siRNA-transfected SW480 cells treated with thymidine for 24h or treated and released from thymidine for 24h. (C) Percentages ofSW480 cells treated with control or Chk1 siRNAs presenting low (1-10 foci/cell) or high (>10 foci/cell) levels of γH2AX foci, or showing pan-nuclear staining for γH2AX during and after exposure to thymidine for the indicated times. Results presented are the means of three independent experiments + standard deviations. (D)) Percentages (%) of γH2AX positive SW480 cells and H2AX+ SW480 cells with a subG1 DNA content following treatment with 2mM thymidine or 2mM HU. Cells were treated with the indicated siRNAs before treatment with the replication inhibitor for 24h before release for 24 or 40h. Cells harvested were analysed for DNA and H2AX content by flow cytometry. Results represent the means of three independent experiments + standard deviations.