Depletion of Pask shows posterior wing margin notching (arrowheads) along with shorter inter-vein distance (see Figure S1b), when co-depleted with mad2 RNAi. Figure S1 b: Quantification of loss of tissue in adult wings: Graph shows the average distance between 4th and 5th vein of adult wings as in S1a, which measures loss of tissue in the engrailed test region. Light grey bars represent candidate RNAi alone, dark grey bars show double knockdowns (candidate RNAi with mad2 RNAi). Negative control (W1118) showed no significant tissue loss with (black bar) or without CIN. The Y-axis starts from 250 mm, to improve resolution. dWNK was an outlier not included in this graph, showing an inter-vein distance without and with mad2 RNAi of 245 mm and 60 mm respectively. Error bars represent 95%CIs, n$8 in all cases. (PDF) Figure S2 a:Quantitation of Acridine Orange staining on larval wing discs. Graph shows quantitation of Acridine Orange staining of control and candidate-RNAi imaginal wing discs. Quantification shows arbitrary grey value units normalized by subtracting the mean grey value of the wild type (anterior) region from the mean grey value of the affected (posterior) region for each disc. Negative control (LacZ RNAi) and candidate RNAi alone are represented in light grey bars and the double knockdowns of candidates with mad2 are represented by dark grey bars, while double knockdown of mad2 with the LacZ negative control is shown in black. Error bars indicate 95%CIs, n$8 in all cases. P-values are calculated by two-tailed t-tests with Welch’s correction: p,0.001 = www, p 0.00120.01 = ww, p 0.0120.05 = w. All t-tests compare candidate-RNAi mad2-RNAi with lacZ-RNAi mad2-RNAi. Figure S2. (b1–b4):Acridine Orange staining on larval wing discs (complete data). All wing discs are stained with Acridine Orange and the dotted line shows the en.CD8GFP marked posterior compartment or test region in which the genes were depleted. The other half of each disc expressed no transgenes. Single knockdowns of each candidate are arranged on the right and the double knockdowns with mad2 are on the left side. Representative discs for each genotype are shown; the level of variation for each genotype can be seen in Figure S2A. (PDF) Figure S3 DNA damage staining quantitation. Graph shows a quantitative analysis of DNA damage (anti-P-H2AvD staining). The Y-axis represents the level of P-H2AvD staining in the affected half normalized by subtracting the level in the control half for each disc. Light grey bars represent the candidate References 1. Mertens F, Johansson B, Mitelman F (1994) Isochromosomes in neoplasia. Genes, Chromosomes and Cancer 10: 221–230. 2. Gao C, Furge K, Koeman J, Dykema K, Su Y, et al. (2007) Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations. Proceedings of the National Academy of Sciences 104: 8995–9000. 3. Heilig CE, Lo¨ffler H, Mahlknecht U, Janssen JWG, Ho AD, et al. (2009) Chromosomal instability correlates with poor outcome in patients with myelodysplastic syndromes irrespectively of the cytogenetic risk group. Journal of Cellular and Molecular Medicine 14: 895–902. 4. Swanton C, Nicke B, Schuett M, Eklund AC, Ng C, et al. (2009) Chromosomal instability determines taxane response. Proceedings of the National Academy of Sciences 106: 8671–8676. 5. Sotillo R, Schvartzman J-M, Socci ND, Benezra R (2010) Mad2-induced chromosome instability leads to lung tumour relapse after oncogene withdrawal. Nature 464: 436–440. 6. Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher MR, et al. (2001) Securin Is Required for Chromosomal Stability in Human Cells. Cell 105: 445– 457. Killing Cells with Chromosomal Instability knockdown in wild type background and dark grey bars represent the double (candidate and mad2) knockdown. Error bars indicate 95%CIs, n$8 in all cases. P-values are calculated by two-tailed t- tests with Welch’s correction: p,0.001 = www, p 0.0120.05 = w and p.0.05 = ns (not significant). (TIF) Figure S4 Cell death assay for the validation of Mad2 results with BubR1. Dotted line shows the en.CD8GFP region or tester region and the other half expresses no transgenes (a-f) Images of wing discs stained with Acridine Orange. (a) mad2 RNAi (d) bubR1 RNAi. (b & e) Pask RNAi. Double knockdowns are (c): Pask RNAi and mad2 RNAi and (f): Pask RNAi and bubR1 RNAi. (TIF) Figure S5 CIN levels. Graph represents the frequency of defective anaphases in knockdowns of LacZ, Asp, Bsk, aPKC, Pask and Mad2 in brain cells. LacZ was used as a negative RNAi control and Mad2 is used as positive control to compare the level of CIN. None of the candidates show significantly elevated levels of CIN above the LacZ control. Error bars show 95%CIs, n.40 in all cases. P-values are calculated by two-tailed Fisher’s exact test: p,0.001 = www (extremely significant) and p.0.05 = ns (not significant). (TIF) Table S1 List of kinases and phosphatases tested in the viability screening of our induced-CIN model. Columns show: the gene identifiers; number of replicate crosses carried out; total numbers of CyO (kinase-RNAi only) and non-CyO (kinase, mad2 RNAi) progeny; Ratio of the average number of Cy/non-Cy progeny per cross used to rank the table; and probability of finding a cross this diverged or more diverged from a 50/50 ratio (the hypothesis), out of this number of crosses (936), purely by chance. (XLSX) Acknowledgments We thank the Bloomington and VDRC stock centers for provision of flies. We thank the University of Adelaide and University of Melbourne for providing facilities and student scholarships. 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Musacchio A, Salmon ED (2007) The spindle-assembly checkpoint in space and time. Nat Rev Mol Cell Biol 8: 379–393. 13. Rieder CL, Maiato H (2004) Stuck in division or passing through: what happens when cells cannot satisfy the spindle assembly checkpoint. Dev Cell 7: 637–651. 14. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JKV, et al. (1998) Mutations of mitotic checkpoint genes in human cancers. Nature 392: 300–303. PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47447