Localization of Top2α to DNA knots on spontaneous chromatin bridges. (A) Experimental procedure to study replication-stress-induced chromatin bridges in BE cells. (B) Schematic of potential mechanisms of spontaneous or replication-stress-induced chromatin bridges. Interlinked cen DNA is shown in red. (C and E) FISH analysis of chromatin bridges in BE cells using a cen probe. DNA bridges (boxed areas) are shown at higher magnification. Small arrows indicate a cen-positive chromatin bridge. (D and F) Frequency of chromatin bridges that are positive for cen or tel probes by FISH. Mean ± SD from three independent experiments (n > 60). (G) Chromatin bridges exhibiting DNA knots next to the midbody. Midbody rings are stained with Cep55 or Mklp1. (H) Localization of GapR:GFP and Top2α to DNA knots on chromatin bridges. (I) Localization of Top2α in cells extracted with the differential retention assay protocol. (J–L) Localization of double-strand DNA ends and mean intensity of TUNEL staining at the bridge DNA next to the midbody in untreated (control) cells or cells treated with Top2α siRNA. (M and N) Localization and mean intensity of Top2α in cells expressing GFP or TDP2:GFP. Mean ± SD, from n cells. Values in control or GFP were set to 1. (O) Frequency of broken chromatin bridges in cytokinesis. Mean ± SD from three independent experiments (n > 120). ***, P < 0.001 (Student’s t test). Arrowheads indicate DNA knots. Midbodies (boxed areas) were reexported and are shown at higher magnification. Numbers below/next to each bar indicate n. Scale bars, 5 μm.