Materials and Methods Stocks All UAS-RNAi lines used, including 485 UAS- Candidate RNAi (kinase and phosphatase) lines, were obtained from the Vienna Drosophila RNAi Centre. The mad2-RNAi line used was v47918. Ubiquitous expression was driven using daughterless (da)-Gal4 and posterior wing disc expression was driven using engrailed (en)- or hedgehog (hh)-Gal4 (Bloomington stock center). Screening In viability screening we have tested ubiquitous (da.Gal4) knockdown of candidates (the kinase and phosphatase genes, see Table S1) in the CIN background (mad2 RNAi): UAS.mad2- RNAi/CyO; da.Gal4/TM6 tub.Gal80ts 6 UAS. candidate- RNAi. The temperature of crosses was adjusted to give the best numbers of progeny while still getting effective knockdown (mostly 23uC). The ratio of the average viable fly count of double knockdown (mad2 and candidate) over single knockdown (candidate only) progeny gave the level of viability of that candidate in CIN flies. Depletion of candidates that gave .75% lethality when mad2 was co-depleted, compared to the candidate knockdown alone, were retested and, if reproducible, considered for further assays. This was measured by counting the number of surviving Cy versus non-Cy progeny from each cross, selecting those with a ratio of at least 4:1. RNA Purification and Quantitative Real-time PCR (qPCR) Assays For each genotype, thirty brains or sixty imaginal wing discs from third instar larvae were dissected in 1X PBS and quickly transferred and homogenized into pre-cooled Trizol reagent (Invitrogen). This was done in three biological replicates. Samples were then frozen in liquid nitrogen and kept in 280 C until RNA extraction. Total RNA was extracted with chloroform and precipitated with ethanol. RNA was further purified using the RNeasy Mini Kit (Qiagen). The qPCR assays were carried out using protocols described in [59]. Each reaction was done in triplicate for all biological replicates. Superscript III (Invitrogen) was used to make cDNA and the relative quantitation was done by using the SYBR Green mix and ABI Prism 7000 Sequence Detection System (Applied Biosystems). The mRNA levels were normalized by the mRNA levels of house-keeping gene ribosomal protein 49 (rp49) from Drosophila. Primers Pairs for Drosophila Mad2, Pask and Bsk qPCR Assay mad2 F/R: GGCGACCAAAAACTGCATCA/GGTAAATT- CCGCGTTGGAAGA, bsk F/R: GAATAGTATGCGCCGCT- TACGA/ATTCCCTATATGCTCGCTTGGC, asp F/R: AGG- CAAAGGCGGTAAACTCTGT/ACTCCGAACACCACATG- AGCAG and (House-keeping gene) rp49 F/R: ATCGATATGCTAAGCTGTCGCAC/TGTCGATACCCTT- GGGCTTG [59]. Histology Loss of tissue in the posterior compartment of adult wings was scored by measuring the distance from where the fifth vein met the margin, to the fourth vein. Wings of adult females were dissected in ethanol and mounted in Aqua Poly/Mount (Polysciences, Inc.). Levels of CIN were tested by counting defective anaphases in fixed, Hoechst 33342 stained brain squashes as described [60]. Killing Cells with Chromosomal Instability Briefly, third instar larval brains were dissected in PBS, fixed for 30 minutes in 4% formaldehyde, then treated with 45% acetate for 30 seconds and put into a drop of 60% acetate for 3 minutes before squashing, freezing in liquid nitrogen and leaving in ethanol until staining with 5 ug/ml Hoechst 33342 in PBS plus 0.2% Tween20, then washing with PBST and mounting in 80% glycerol. All clear anaphases in each brain were photographed and scored as normal or defective. Defective included those with bridges, broken bridges or lagging chromosomes. Slides were coded and scored without knowing their genotype. Cell death in 3rd instar larval imaginal wing discs was measured using a vital stain (Acridine Orange). Third instar larvae were selected and dissected, and imaginal discs were collected carefully in PBS. Imaginal wing discs were then incubated for 2 min in a 1 mM Acridine Orange solution and briefly rinsed in PBS before immediately mounting and imaging. The discs were transferred to a slide having double sided sticky tape on either side of the sample. This was done to prevent the squashing of discs when we placed a cover slip on top. Acridine staining was normalized by subtracting the mean intensity value of the wild type anterior compartment from the mean intensity value of the mutant posterior compart- ment (marked with en.CD8GFP), using ImageJ software. Before normalization, background noise was subtracted from all the images by setting the rolling ball radius at 10 pixels. Cleaved caspase 3 immunostaining was performed on dissected wing imaginal discs. Single and double knockdown third instar larvae were selected and dissected in PBS and fixed in 4% formaldehyde for 20 minutes. Fixed discs were extensively washed with PBST (1xPBS+0.2% Tween) and then blocked by PBST containing 5% fetal calf serum (PBSTF) for 30 minutes. Discs were then incubated in primary antibody solution (1:100 Cleaved Caspase 3 Antibody from Cell Signaling in PBSTF) for 2.5 hrs at room temp or left overnight at 4uC followed by 2–3 quick washes with PBSTF and then 30 minutes in PBSTF. For secondary antibody staining, discs were incubated for 2 hrs in the dark at room temperature with 1:75 Anti rabbit CY-3 Antibody from Abacus/Jackson in PBST followed by 2–3 quick washes with PBST and then 30 minutes incubation in PBST. Mounting was done with 80% glycerol-PBS. DNA damage staining was done using the same method with rabbit anti-H2AvD P-Ser137 (1:700; Rockland) which is the Drosophila equivalent of vertebrate cH2aX, and anti-rabbit Cy3-conjugated secondary antibody (1:100; Abacus/Jackson). Quantitation of DNA damage staining (anti-P- H2AvD) was also done on normalized mean intensity value. Normalization was done by subtracting the mean intensity value of the wild type anterior compartment from the mean intensity value of the mutant posterior compartment (marked with en.CD8GFP), using ImageJ software. Before normalization, background noise was subtracted from all the images by setting the rolling ball radius at 5 pixels. All microscopy was done on a Zeiss Axioplan2 with Semrock Brightline filters and measurements and quantitation were done using Axiovision software (Carl Zeiss). Images were compiled using Axiovision (Carl Zeiss), Photoshop and Illustrator (Adobe) software. Supporting Information Figure S1 a. Loss of tissue in adult wings. Engrailed-driven single (candidate-RNAi only) and double (candidate and mad2-RNAi) knockdown in adult Drosophila wings. This driver depleted genes only in the posterior compartment of the wing, the lower half of each wing in this figure. We measured the loss of tissue by measuring the shortest distance from where the fifth longitudinal vein met the margin, to the fourth longitudinal vein (arrows). PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47447