Fig. S1. Control Reh and U698 cells were stained for γH2AX and DNA content as described in “Materials and methods” (upper panels), or by leaving out the primary antibody (“staining control”; lower panels).

G1

G2L

G2H+MS

G1

G2L

G2H+MS

Fig. S2. Peripheral blood B lymphocytes from a healthy donor stimulated to enter the cell cycle. Cells were stained for γH2AX and DNA content as described in “Materials and methods”.

EdU (Alexa Fluor 647)DNA content (Hoechst 33258)

Phos

pho-

Hist

oneH

3 (P

E)γH

2AX

(FIT

C)TU

NEL

(Per

CP-C

y5.5

)

G2 Low

G2 High

S

G1

M

Non-apoptotic cells

Non-mitotic cells

Fig. S3. Gating strategy used for determining the cell cycle-resolved fractions of EdU-positive cells (to the left of dotted line), based on γH2AX and DNA staining after removal of aggregates (not shown), and apoptotic cells (upper left panel). This example shows U698 control cells 3 h after a 10 min EdU pulse (10μM). The same gates were applied for sorting followed by counting of the γH2AX foci by microscopy (results in Fig 2B).

Reh U698

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

JVM-2C

ontr

olP

AR

Pi t

reat

ed

Fig. S4. Uncorrected fractions of EdU+ cells in different phases and compartments (raw data). The spillover of S phase cells, assessed at time zero, was particularly pronounced into G2H, less so into G2L, and almost zero into M and G1, with gates as shown in Fig. S3. This spill over was compensated for in Fig. 3 as described in “Materials and methods”.

Fig. S5. (A) The predicted time course (normalized to experimentally determined maximum labelled fraction) of EdU+ cells with fixed durations of G2H, G2L and M (5, 2.5 and 0.5 h, respectively), and sequential movement through the 3 compartments. (B) The predicted time course with durations of G2H 0.5-7.5h (flat distribution), while G2L and M had lengths of 2.5 and 0.5 h. (C) As in (B), but with 32% of the cells entering G2L directly from S phase (f(0)=0.32). Experimental data are shown for control U698 cells.

A B C

G2H; H2AX high

G2L; H2AX low

Mitosis G1

Simulated G2H; H2AX high

Simulated G2L; H2AX low

Simulated Mitosis

U698 5 2.5 0.5

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

U698 0.5-7.5 2.5 0.5

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

U698 0-7.5 (f(0)=0.32) 2.5 0.5

Time (hours)

0 2 4 6 8 10 12 14 24

% E

du+

0

20

40

60

80

100

# foci

0 10 20 30 40 50 60

# cells

0

2

4

6

8

G2H

# foci

0 10 20 30 40 50 60

# cells

0

5

10

15

20

25

30

35

G2L

Reh

U698

# foci

0 10 20 30 40 50 60

# cells

0

2

4

6

8

10

12

G2L

# foci

0 10 20 30 40 50 60

# cells

0

2

4

6

8

10

G2H

Fig. S6. γH2AX focus numbers in sorted control G2H and G2L cells (see Fig. S3 for strategy and sort gates). Focus numbers for mid-S phase cells are shown for comparison. The means and standard deviations of these distributions are given in Fig.2B.

# foci

0 10 20 30 40 50 60

# cells

0

2

4

6

8

10

12

14

16

18

S

# foci

0 10 20 30 40 50 60

# cells

0

2

4

6

8

10

12

14

16

S