Supplementary Figure 1: Chromosome segregation and abscission dynamics during replication stress.

(a) Wild type cells expressing Htb2-mCherry were treated with 100 mM HU for 2 h, and transferred to fresh media. The Htb2-mCherry signal was used to estimate the frequency of anaphase (with elongated nuclei) or telophase (with separated nuclei) cells at the indicated times (left). Two independent experiments were performed with similar results; results from one experiment are shown. The following number of cells n was scored for each time point: n = 170 (0 min), 146 (30 min), 160 (60 min), 156 (90 min), 164 (120 min), 124 (150 min), 149 (180 min). The micrographs show cells 120 min after HU washout stained with DAPI to visualize DNA (right). The arrows point to chromatin bridges. (b) Kinetics of nuclear division (visualized with Nsg1) relative to actomyosin ring contraction (Myo1) in wild type cells in log-phase or after exposure to HU at 30 °C, as in Fig. 2a. Asterisk marks the time of nuclear division. Numbers indicate time in minutes. The graph shows the time of nuclear division relative to the onset of ring contraction (time 0). Boxes include 50% of data points, whiskers 90%; medians are shown as lines and means as crosses. The kinetics between the two conditions were considered to be not statistically significant (P > 0.05, Mann-Whitney test). n = 32 untreated cells and 30 HU-treated cells, pooled from two independent experiments. (c) The time of chromosome segregation (Htb2-mCherry) relative to actomyosin ring contraction (Myo1-GFP) in wild-type cells in the absence or presence of 60 mM HU. HU-treated cells were imaged for 2 h immediately after HU addition. The graph shows the time of chromosome segregation relative to the onset of ring contraction (time 0). Median (lines) and mean (crosses) are shown (n = 80 untreated cells and 66 HU-treated cells, pooled from 3 independent experiments). (d) Segregation of the rDNA (Net1-GFP) and TEL12R (TetR-YFP) in a representative wild-type cell in the presence of 60 mM HU, as in (c). Arrowheads mark the spindle poles, an asterisk marks the nucleolus, and arrows mark TEL12R. Time is shown in minutes relative to anaphase onset; nucleolar segregation occurs at 10 min and TEL12 segregation follows at 12 min. The graph shows the time of segregation of the rDNA and TEL12R in the absence and presence of HU. n = 50 untreated and 42 HU-treated cells, pooled from 2 independent experiments (P > 0.05, Student’s t-test). (e–h) Abscission dynamics of untreated and HU-treated wild type cells (d), sml1Δ (e) and ahc1Δ (f) mutant cells, imaged as in (c). The graphs in (e,f) show the fraction of cells completing abscission relative to the time of membrane ingression; the median abscission times are shown in (h). WT and WT + HU from (d) are represented as dashed lines for comparison in (f–g). n is the total number of cells in each category, pooled from two independent experiments, and is shown in brackets in (h). Asterisk marks significant differences between WT treated and untreated cells (P < 0.0001, Mann-Whitney test). In (b–d), boxes include 50% of data points, whiskers 90%; medians are shown as lines and means as crosses. Scale bars in (a,b,d): 2 μm.