Topo 2 assay kit

DPBQ and other chemicals were obtained from the National Cancer Institute, diluted in stock DMSO solutions and used at the concentrations shown. Other chemicals used include doxorubicin (ThermoFisher, Waltham MA), etoposide (Topogen, Port Orange, FL), paclitaxel (ThermoFisher/Acros), BI-2536 (Selleck Chemical, Houston, TX), chloroquine, 17-AAG, and Nutlin-3a (Fisher). Antibodies used include β-actin (ab6276 WB 1:10,000; Abcam,Cambridge, MA), pericentrin (ab4448 IF 1:1000; Abcam), p53 (#9282, WB 1:2000; Cell Signaling Technology, Beverly, MA), phospho-P15 p53 (#9284 WB 1:2000; Cell Signaling Technology), p21 (sc6246, WB 1:500; Santa Cruz, Dallas, TX)
The topoisomerase assay was performed using the Topo II Assay kit per the manufacturer instructions (TopoGEN, Port Orange, FL). Circular dichroism of 0.5 mg/ml salmon sperm DNA was performed using an AVIV Model 420 CD Spectrometer at room temperature. To analyze DNA binding, 100 μM ethidium bromide or DPBQ was added to the sample to evaluate changes in spectrum consistent with intercalation.

DNA Topo II activity was measured by decatenating kinetoplast DNA (kDNA) with a Topo II assay kit (TopoGEN, Port Orange, FL). In brief, equal volumes of buffers A (0.5 M Tris-HCl, pH=8, 1.5 M NaCl, 100 mM MgCl₂, and 5 mM dithiothreitol) and B (20 mM ATP in water) were mixed to make a fresh 5 × complete assay buffer. Each 20 μl of reaction mixture contained 4 μl of 5 × complete assay buffer, 200 ng of kDNA, and 100 ng (or 300 ng) of nuclear extract. To determine whether Topo II activity was regulated by MIIP specifically, we used a mixture of 0.25 IU of Topo IIα (TopoGEN) and different doses of purified MIIP protein (OriGene, Rockville, MD) to replace the nuclear extracts. This reaction was kept at 37° C for 30 min and stopped with 4 μl of 5×Stop buffer. The mixture was loaded in 1% agarose gel and run for 1 h at 50 V by electrophoresis. The catenated and decatenated kDNA in the gel was stained with ethidium bromide and photographed under ultraviolet illumination. Topo II activity was quantified on the basis of the amount of decatenated kDNA using Image J software from the National Institutes of Health website (http://rsb.info.nih.gov/ij/).