In vitro Topo II activity assay using agarose gel electrophoresis and western blotting.

(A) Approximately 0.25 μg pBR322 DNA was incubated with 1 unit of human Top IIα and 1.6 μM HMNE3 for 30 min at 37°C in a total of 20 μL of reaction buffer. The reaction was then stopped with 2 μL of 10% SDS and 1 μL proteinase K. The samples were subjected to electrophoresis on a 0.8% agarose gel in 1× TAE at 5 V/cm for 1 h. Then, the gel was stained with 0.5 μg/mL of ethidium bromide (EB) for 30 min, destained with distilled water for 30 min, and photographed under a UV trans-illuminator. (B) The expression levels of Top IIα and Top IIβ were detected by western blotting.