1. Figure S1 A
MRC5
Control
Chk1
TdR(h):
γH2AX
Cleaved casp3
RPA34
β-actin
siRNA: B
SW480
Control
Chk1
TdR(h):
γH2AX
Cleaved casp3
RPA34
β-actin
siRNA: C
HCT116
TdR(h):
γH2AX
Cleaved casp3
RPA34
Chk1
β-actin - +
Chk1 inhibitor (Gö6976) 0.1 mM
10Gy
Figure S1. gH2AX 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.

2. Chk1
Control
Time(min)
after IR
(10Gy):
γ-H2AX
RPA34
β-actin
siRNA:
Figure S2 A
Control HCT116 cells B
Min post IR
γH2AX
RPA34
Merge
γH2AX
RPA34
Merge
Min post IR
Chk1 depleted HCT116 C
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 gH2AX 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.

3. Figure S3
Time(h) in thymidine 5 10 15 20 25 30 35 40 45 50 60
% cells cleaved casp-3
Chk1-depleted -TdR
Chk1-depleted +TdR
Control-depleted -TdR
Control-depleted +TdR A
% TUNEL + cells 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.

4. HCT116 Chk1-depleted cells
DNA
γH2AX
merge II I
III
I (1-10 foci)
II (>10 foci)
III (diffused
pan nuclear staining)
TdR (h): 6h
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.

5. Figure S5 A C
SW480 B
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.

6. Figure S6 A B
Gated R3 region cells
siRNA:
control
Chk1
Cdc45
Chk1/Cdc45
control
Chk1
Cdc45
Chk1/Cdc45
DNA content
untreated
6.5%
5.2%
5.6%
10.9%
γH2AX
DNA content
24hTdR
11.3%
58.3%
9.1%
14.4%
control
Chk1
Cdc45
Chk1/Cdc45
DNA content
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).

7. Figure S7
Hours in TdR
control
Chk1 A 24
Continuous exposure to TdR(h)
Release in TdR-free medium (h)
control
Chk1 32 40 48 64 8 16 24 40 24
Hours in TdR 0%
20%
40%
60%
80%
100%
control
Chk1
G2/M S G1
SubG1 32 40 48 64
Continuous exposure to TdR(h)
Release in TdR-free medium (h) 8 16 B
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.

8. Figure S8
untreated
2.7%
Control siRNA
1.4%
40hTdR
4.6%
31.2%
3.2%
24.1%
DNA content
Active casp-3
Chk1 siRNA
24hTdR,
24h Release
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.

9. Figure S9 A
Control siRNA B
Control siRNA
6h, R40h
16h, R30h
46h
30h 6h
16h 0h 0h
Chk1 siRNA
Chk1 siRNA 0h 6h
46h
6h, R40h
16h, R30h
30h
16h 0h
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.

10. Figure S10 A B SW480 24h thymidine Control siRNA Chk1 siRNA
h post BrdU pulse
G1: 14.4
S: 64.1
G2: 10.73
89.3%
G1: 6.2 S:
G2:
88.6%
G1: 34.9 S:
G2:
86.7%
G1: 10.9
S: 22.9
G2:
36.2%
G1: 5.7 S:
G2:
37.4%
G1: 10.2
S: 21.4
G2: 11.54
43.2%
Total cells B
SW h HU
Control siRNA Chk1 siRNA
h post BrdU pulse
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 mM 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).

11. - - + + - - - + + - - - - - - + + - - - + + + - - - - - - - - - -
Figure S11
SW480 A
TdR(24h):
HU(24h)
IR(10Gy)
Release(24h)
Chk1
pSer1981 ATM
Control
siRNA:
pThr68 Chk2
β-actin
γH2AX
RPA34 B
Control siRNA
Chk1 siRNA
Release (h):
TdR (h):
γ-H2AX
% cells with H2AX foci
untreated
TdR24h
R24h
Control
Chk1
siRNA:
1-10 foci
>10 foci
pan nuclear 10 20 30 40 50 60 C D 10 20 30 40 50 60 70 80 90
100
TdR(h):
Release (h):
Control
Chk1
siRNA:
H2AX+
H2AX+ with SubG1
% cells
Release. (h):
HU(h):

12. Figure S11. Persistence of gH2AX 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 gH2AX+ 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.