In vitro of complex (ICE) Assay

Topoisomerase II DNA-protein complexes (TOP2-DPCs) were isolated and detected using in vivo complex of enzyme (ICE) assay as previously described (Anand et al., 2018). Briefly, 5 million cells were lysed in sarkosyl solution (1% w/v) after treatment. Cell lysates were sheared through a 25 g 5/8 needle (10 strokes) to reduce the viscosity of DNA and layered onto CsCl solution (150% w/v), followed by centrifugation in NVT 65.2 rotor (Beckman coulter) at 42,000 RPM for 20 hr at 25°C. The resulting pellet containing nucleic acids and TOP2-DPCs was obtained and dissolved in TE buffer. The samples were subjected to immunoblotting (slot blot) with anti-mouse TOP2β antibody (Novus). 2 μg of DNA is applied per sample. TOP2-DPCs were quantified by densitometric analysis using ImageJ.