Sc 32736

TOP1 protein–DNA complexes (TOP1cc) were purified using caesium chloride density gradients. Cells (2 × 10⁶) were lysed in 1% sarcosyl, 8 M guanidine HCl, 30 mM Tris pH 7.5 and 10 mM EDTA. Cell lysates were incubated at 70°C for 15 min to remove all non-covalently bound proteins from DNA. Cell lysates were then loaded on a caesium chloride density (CsCl) step gradient (5 ml total volume) and centrifuged at 75 600 × g at 25°C for 24 h to separate free proteins from DNA. Ten consecutive 0.5 ml fractions were collected and slot blotted onto Hybond-C membrane (Amersham). To ensure equal DNA loading, the DNA concentration in each extract was determined fluorimetrically using PicoGreen (Molecular Probes/Invitrogen). Covalent TOP1–DNA complexes were then detected by immunoblotting with anti-TOP1 antibodies (sc-32736, Santa Cruz) and visualised by chemiluminescence. Fractions enriched for TOP1cc were pooled and subjected to serial dilution followed by band quantifications using the BioRad ChemiDoc platform.

Commercial human Topo I siRNA was used in this study (human Top1 siRNA, sc-36694; Santa Cruz Biotechnology). TOP1 was analysed by western blotting using a mouse anti-Topo I antibody (C-21) (1:10,000, sc-32736; Santa Cruz Biotechnology). Commercial human MUS81 siRNA was used in this study (human MUS81 siRNA, L-016143-01-0005; Horizon). MUS81 was analysed by western blotting using a mouse anti-MUS81 antibody (MTA30 2G10/3) (1:10,000, ab-14387; Abcam). As a loading control, α-tubulin was analysed with mouse anti-α-tubulin antibody (10G10) (1:10,000, 017-25031; Fujifilm). Horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (1:10,000, 715-035-150; Jackson ImmunoResearch Laboratories) and HRP-conjugated anti-rabbit IgG (1:10,000, 711-035-152; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. The bands were visualised with Chemi-Lumi One Super (Nacalai Tesque) and analysed with LAS4000 Mini (GE Healthcare Life Science).

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).