Fig. 8. Segmentation of individual replication domains (RDs), pulse-labeled in early S-phase, indicate structural changes after cohesin depletion.

a SIM nuclear mid-sections of nuclei with typical early S-phase patterns of replication domains (RDs) from a control culture (left), a 6 h auxin-treated culture (middle), both fixed 6 h after RL, and a multilobulated nucleus (right) obtained after 30 h auxin treatment (compare Fig. 6a–d). Enlargements of boxed areas show individual, segmented RDs displayed in red with segmented borders lined in yellow. Scale bar: 4 µm, 0.5 µm in inset magnifications. b Counts of segmented RDs plotted for control nuclei, cohesin depleted nuclei after 6 h auxin and MLN after 30 h auxin are presented as dots (control: n = 11, 6 h: n = 7, 30 h: n = 11 nuclei from two independent experiments). c Boxplots with corresponding volume distributions of segmented individual RDs (control: n = 39.334, 6 h: n = 31.467, 30 h: n = 55.153). The non-parametric two-sided Mann–Whitney test revealed significant differences of RD counts between control nuclei and MLN (p = 0.012) and for RD volumes (p < 0.0001 for control<-> 6 h auxin, control<−> 30 h auxin, and 6 h auxin<−> 30 h auxin). Volumes of RDs with dimensions below the resolution limit of 3D-SIM (~120 nm lateral/300 nm axial) show the same size and were excluded from consideration. Accordingly, the lower limits of volumes between control nuclei and cohesin depleted nuclei are identical in contrast to the differences of the upper volume limits. Data in b, c are represented as boxplots where the middle line indicates the median, the lower and upper hinges correspond to the 25 and 75% quartiles, the upper whisker extends to the largest value no further than 1.5 × IQR (inter-quartile range) from the hinge and the lower whisker extends to the smallest value from the hinge at most 1.5 × IQR. In b, all data points are plotted individually. Source data are provided as a Source Data file.