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.