Top2α localizes to DNA knots on HU-induced chromatin bridges. (A–C) FISH analysis of spontaneous or HU-induced chromatin bridges in BE cells using a tel or cen probe. DNA bridges (boxed areas) are shown at higher magnification. (D–F) Localization of FANCD2. In F, cells were treated with 1 mM HU for 2 h. (G) A DNA knot from a spontaneous bridge. Small arrows indicate thin DNA strands at the DNA knot. Scale bar, 1 μm. (H and I) Frequency of chromatin bridges exhibiting DNA knots next to the midbody. Mean ± SD from three independent experiments (n > 150). (J–L) Localization of GapR:GFP, Top2α, and Top2β. (M) Mean intensity of Top2α at the bridge DNA next to the midbody in the absence (control) or presence of Top2α siRNA. Values in control were set to 1. (N) Localization of Top2α by using a different polyclonal antibody against Top2α. (O) Mean intensity of Top2α at DNA knots on spontaneous chromatin bridges exhibiting relatively strong or weak DNA staining at the midbody. In cells with chromatin bridges, the mean fluorescence intensity of DNA at the midbody was set to 1. Midbody DNA-fluorescence intensity >1 was taken as strong whereas midbody DNA-fluorescence intensity <1 was classified as relatively weak. The mean intensity of Top2α at the midbody in cells with strong or weak midbody DNA signal was plotted. Values in strong DNA signal were set to 1. (P) Mean intensity of Top2α at the bridge DNA next to the midbody on chromatin bridges of various lengths. Mean ± SD from n cells. Values in <20 μM were set to 1. ***, P < 0.001 (Student’s t test). Numbers below/next to each bar indicate n. (Q) Localization of KIF4A. Arrowheads indicate DNA knots. Midbodies (boxed areas) were reexported and are shown at higher magnification. Scale bars, 5 μm.