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). G1G1 G2LG2L G 2 H+M S G1G1 G2LG2L S

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) Phospho-HistoneH3 (PE) γH2AX (FITC) TUNEL (PerCP-Cy5.5) G 2 Low G 2 High S G1G1 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).

RehU698JVM-2 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 G 2 H, less so into G 2 L, and almost zero into M and G 1, 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 G 2 H, G 2 L 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 G 2 H h (flat distribution), while G 2 L and M had lengths of 2.5 and 0.5 h. (C) As in (B), but with 32% of the cells entering G 2 L directly from S phase (f(0)=0.32). Experimental data are shown for control U698 cells. ABC

Reh U698 Fig. S6. γH2AX focus numbers in sorted control G 2 H and G 2 L 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.