Studying Anticancer Drug Properties in Drosophila Figure 4. Evidence for F14512-specific Topo II-mediated cleavage in moderately repeated Drosophila sequences. (A) HindIII-digested DNA samples from S2 cells treated for 30 minutes with no drug (lanes 1and 7), F14512 (5, 10, 25 or 50 mM, lanes 2–6 and 9–12) or 50 mM TOP-53 (lanes 2 and 8), in the absence (lanes 1–7) or presence (lanes 8–12) of 25 mM distamycin (DIST). Purified DNA samples were hybridized to a labeled HinfI-digested whole genomic DNA probe that had been pre-hybridized as described in Materials. The a–b–c fragments indicated to the right of the gel are described in the text. (B) Densitometric scan of the autoradiograph shown in (A) for lanes 7, 8 and 11. Regions corresponding to fragments a, b and c are indicated by arrowheads. (C) S2 cells were treated for 30 minutes with no drug (-), F14512 (25 or 50 mM) or 50 mM etoposide in the presence of 25 mM distamycin (DIST). Purified DNA samples were processed as described in panel A. Fragments a, b and c are indicated by arrowheads to the left of the gel, which provides better resolution of the a–b regions. doi:10.1371/journal.pone.0023597.g004 (dispersed) to moderately repeated (tandem or dispersed) sequenc- es (Cot2 DNA) allowed to proceed overnight before stringent washing and exposure to film (12 to 24 hours) as described above. The resulting signal that can be observed (Figures 4A and 4C) corresponds to the genome-wide pattern of moderately repeated HindIII fragments or of dispersed (non-tandem) highly repeated HindIII fragments. The slow kinetics of DNA hybridization onto a solid medium like the nylon membrane (rather than in solution) effectively largely suppress the visualization of single-copy sequences, which otherwise would produce a smear obscuring the distinct pattern of repeated HindIII fragments. Drug treatments and analysis of cytotoxic effects Cell viability (Figure 5A) was measured in response to exposure to F14512 and reference Topo II inhibitors using the method of viable dye exclusion by living cells. Briefly, 2.56106 Kc cells were seeded in a 12-well plate 24 hours before addition of F14512 or the reference Topo II inhibitors. The number of viable cells was then determined 24, 48, 72, and 96 hours post-treatment using the Vi-CELL analyser (Beckman Coulter). The percentage of viable cells was then calculated with reference to the control culture exposed to the drug vehicle 0.1% DMSO, and normalized to 100% for each time point explored. For observation of drug-mediated cytotoxic effects (Figures 5B and 5C), Kc cultures were treated with teniposide, etoposide, TOP-53 or F14512 at the concentrations indicated in the text for up to 72 hours. Live cultures were monitored by phase-contrast microscopy starting at 2 hours and photographs taken using the 40x objective of a Nikon Eclipse TS100-F microscope equipped with a Spot Insight Color CCD camera. For examination of nuclei, aliquots of cells were withdrawn and fixed in PBS containing 0.1% Triton X-100 and 4% paraformaldehyde for 10 minutes at room temperature. Fixed cells were transferred to a microscope slide, the excess fixative carefully removed with a Kimwipe and the nuclei stained with DAPI (10 mg/ml) in anti-fade solution [36]. Coverslips were sealed onto the slides with nail PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23597