Top2α forms Top2–DNA adducts on DNA knots on HU-induced chromatin bridges. (A and B) Localization of PICH and TopBP1 in BE cells. (C) Localization of Top2α in cells extracted with the differential retention assay protocol or the standard fixation protocol for immunofluorescence, in the absence of treatment (untreated) or after treatment of cells with 10 μM etoposide for 2 h. (D) Localization of Top2α on HU-induced chromatin bridges in cells extracted with the differential retention assay protocol. (E and F) TUNEL staining on spontaneous or HU-induced chromatin bridges. (G) A spontaneous bridge with a DNA knot in HeLa cells. (H) Frequency of chromatin bridges that are positive for cen or tel probes by FISH in HeLa cells. Mean ± SD from three independent experiments (n > 60). (I and J) Localization of double-strand DNA ends (TUNEL) and Top2α:GFP at the bridge DNA next to the midbody in HeLa cells. Midbodies (boxed areas) were reexported and are shown at higher magnification. (K) Mean intensity of TUNEL staining at the bridge DNA next to the midbody in BE cells expressing GFP or TDP2:GFP. Mean ± SD from n cells. Values in GFP were set to 1. Numbers below/next to each bar indicate n. (L) Western blot analysis of total GFP, TDP2, and tubulin in BE cells expressing TDP2:GFP. (M) Western blot analysis of total Top2α, Mre11, and tubulin in untreated (control) cells or cells transfected with Top2α siRNA. (N) Frequency of broken DNA bridges in HU-induced cells. (O) BE cells exhibiting Lamin A bridges in the absence (control) or presence of Top2α siRNA. (P) Frequency of broken Lamin A bridges. Mean ± SD from three independent experiments (n > 120). ***, P < 0.001 (Student’s t test). Broken chromatin bridges are indicated by dotted arrows. Arrowheads indicate DNA knots. Scale bars, 5 μm. Source data are available for this figure: SourceData FS2.