Studying Anticancer Drug Properties in Drosophila Figure 3. Cellular profiles of drug-induced Topo II-mediated cleavage in SAT III repeats of the Drosophila X chromosome. (A) Structure of SAT III repeats. 359-bp SAT III repeats accommodate 2 nucleosomes per repeat. One of the two nucleosomal linkers located in each repeat contains a short GC-rich sequence that serves as a highly specific target for Topo II cleavage in vivo, as denoted by scissors. (B) Schneider S2 cells were treated for 30 minutes with teniposide (VM26), etoposide (VP16) or TOP-53 at the concentrations shown, in the absence (-) or presence (+) of distamycin (DIST, 25 mM). Purified DNA was electrophoresed without prior restriction enzyme digestion, transferred to a nylon membrane and hybridized to a labeled SAT III probe. 359-bp: the SAT III monomer (SAT) used as a probe; M: molecular weight standards. (C) S2 cells were treated for 30 minutes with no drug (0, lane 13), F14512 (5, 10, 25 or 50 mM, lanes 14–17) or 50 mM TOP-53 (lane 18), in the presence of 25 mM distamycin (DIST). Omission of the latter gave identical results (not shown). DNA samples were processed as described above. SAT: the 359-bp cloned fragment used as a probe; M: molecular weight standards. doi:10.1371/journal.pone.0023597.g003 exposed to Kodak BioMax MS-1 film with BioMax MS intensifying screens. Exposure times ranged between 0.5 and 14 hours. The procedure used for the global detection of Topo II- induced cleavage in S2 cells is detailed below. Global shotgun search for specific F14512-induced chromatin cleavage DNA from S2 cells treated with Topo II poisons in the absence or presence of distamycin was prepared as described above. Purified DNA samples (1 mg) were digested with HindIII and electrophoresed on 1.2% (Figure 4A) or 0.8%. agarose/TBE gels (Figure 4C). After transfer to GeneScreen Plus membranes, pre- hybridization was performed exactly as described [37] for 2 hours at 42uC in the presence of sonicated and denatured Kc genomic DNA (50 mg/ml) together with sonicated, denatured salmon sperm DNA carrier. During the pre-hybridization step, HinfI- digested Kc genomic DNA (200 ng) was radiolabeled using a random-priming kit (Perkin Elmer) and a-32P-dATP (3000 Ci/ mmole, GE Healthcare). The labeled whole genomic probe was denatured by boiling for 10 minutes and incubated with 10 mg of cold sonicated (average size ,1 kb) S2 genomic DNA in 800 ml of 0.12 M sodium phosphate, 1 mM EDTA. Under these conditions, determined empirically using known Cot curves of Drosophila genomic DNA, incubation at 68uC for 35 minutes results in the rehybridization of snap-back DNA (simple repeats widely spread in the Drosophila genome) as well as of Cot1 DNA (very high- abundance tandemly repeated sequences). Control experiments have shown that these conditions suppress .80% of the normally observed SAT III cleavage ladder. The pre-hybridized probe was added to the nylon membrane and hybridization of highly PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23597