Killing Cells with Chromosomal Instability Figure 2. Cell death assays on larval wing discs. Dotted line shows the en-CD8GFP marked compartment or tester region in which genes were depleted. The other half of each wing disc expressed no transgenes and serves as an internal control. (a–c, a9–c9) Images of wing discs stained with Acridine Orange to show cell death. (a) Negative control (lacZ RNAi), (a9) LacZ and mad2 RNAi, (b & c) Candidate RNAi (asp and bsk), (b9 & c9) double knockdown of candidate and Mad2. (d) Graph shows quantitation of Acridine Orange staining (above wild type) in control and candidate imaginal wing disc halves with or without mad2 RNAi. Error bars represent 95% CIs, n$8 in all cases. P-values were calculated by two-tailed t-tests with Welch’s correction: p,0.001 = www. (e-e9) Cleaved caspase 3 staining showing apoptosis in e: mad2 RNAi, e9: asp RNAi and double knockdown (e0: asp RNAi and mad2 RNAi). doi:10.1371/journal.pone.0047447.g002 plausible hypothesis to explain this sensitivity was that the centrosome disruption by itself caused a certain rate of CIN, which when added to the CIN from Mad2 depletion, took the cells over a threshold of instability beyond which they were unviable [36]. Our data do not support this model, as we did not detect a significant rate of anaphase errors when any of our centrosomal candidates were depleted by themselves (Figure S5). An alternative explanation is that there is significant crosstalk between events at PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47447