Figure 5: Failed cytokinesis triggers RS accompanied by progressive DNA breakage.

(a) A schematic depiction of cellular paths to ploidy increase after failed cytokinesis. (b) Representative image galleries (n=10) of U-2-OS cells stably expressing mRFP-53BP1 treated with the indicated siRNAs and analysed by time-lapse microscopy. Scale bar, 10 μm. (c) Representative image galleries (n=10) of U-2-OS cells stably expressing GFP-PCNA treated with the indicated siRNAs and followed by time-lapse microscopy. Numbers indicate time (h) after siRNA transfections. Arrowheads mark a cell undergoing re-replication. Scale bar, 10 μm. (d) Representative image galleries (n=10) of U-2-OS cells treated and analysed as in (c). Numbers indicate time (h) after siRNA transfections. Arrowheads mark nuclei undergoing asymmetric DNA replication. Scale bars, 10 μm. (e) Representative images (n=100) of U-2-OS cells treated with the indicated siRNA and immunostained for 53BP1. Nuclear DNA was counterstained by DAPI. Scale bar, 10 μm. (f) Representative images (n=100) of U-2-OS cells treated as in (e) and immunostained for γ-H2AX. Scale bar, 10 μm. (g) Representative images (n=25) of U-2-OS cells treated with the indicated siRNA and pre-incubated with BrdU for 5 h before fixation and immunostaining for 53BP1. Insets show × 2 magnification of the indicated areas. Scale bar, 10 μm. (h) Representative images (n=25) of U-2-OS cells treated with the indicated siRNA and immunostained for 53BP1 and γ-H2AX. Nuclear DNA was counterstained by DAPI. Arrowheads mark the fragmented nuclei that lost 53BP1 and where DSBs are decorated solely by γ-H2AX. Scale bar, 10 μm. (i) Representative images (n=25) of U-2-OS cells stably expressing Gam-GFP treated with the indicated siRNA and immunostained for Lamin A and γ-H2AX. Nuclear DNA was stained by DAPI. Insets show 2x magnification of the indicated areas. Scale bar, 10 μm.