RADAR Assay for DNA Damage Detection

RADAR assay was performed as in (46 (link)) with minor modifications. Approximately 5 × 10⁵ cells were treated with PDS, CPT, ETO or CX-5461 (as above) for 1 h. After drug treatment, medium was aspirated and cells were lysed directly on the plate by adding 1 ml of lysis reagent (RLT Plus, Qiagen). Nucleic acids were recovered by adding 0.5× volume of 100% ethanol, incubation at -20°C for 10 min and then centrifugation at maximum speed at 4°C for 15 min. The supernatant was removed and the pellet was washed twice with 75% ethanol, followed by 10 min centrifugation at maximum speed. The nucleic acid pellet was then dissolved in 200 μl of freshly prepared 8 mM NaOH and rotated overnight at 4°C to ensure complete solubilisation.
Samples were then slot blotted onto PVDF membranes or Zeta-probe membranes which were then probed for using standard Western blotting as above. Antibodies used were mouse anti-TOP1 (Santa Cruz, sc-32736, 1:200), mouse anti-TOP2A (Santa Cruz, sc-365916, 1:200), mouse anti-dsDNA (Abcam, ab27156, 1:10 000).