TopoIIα extraction and decatenation assay

HEK293 cells were induced to express GFP-TopoIIα for 24 h while endogenous TopoIIα was depleted with siRNA. Cells were twice pelleted and resuspended in ice-cold TEMP buffer (10 mM Tris pH7.5, 1 mM EDTA, 4 mM MgCl₂, 0.5 mM PMSF) before being left on ice for 10 min. Lysates were homogenised by ten strokes through a 25 G needle before nuclei were pelleted by centrifugation (1500 × g, 10 min). The nuclear pellet was resuspended in TEMP buffer and washed, then resuspended in 4 pellet volumes of TEP buffer (10 mM Tris pH7.5, 1 mM EDTA, 0.5 mM PMSF) and an equal volume of 1 M NaCl added. Preparations were vortexed and left on ice for 60 min. Finally, nuclear preparations were centrifuged at 4 °C for 15 min at 15,000 × g. Supernatants were diluted with 4X NuPAGE LDS sample buffer and immunoblotted for TopoIIα levels. TopoIIα levels were equalised prior to use in the TopoGEN kinetoplast DNA assay as above.