in-house, were preferentially used in this study. Briefly, dsRNA were validated in quadruplicate, with one set of plates treated with MMS and one set used as an untreated control. Plates with or without MMS with the same dsRNA were then compared. Genes required after MMS treatment and genes whose knock- down conferred resistance to MMS were validated by validation amplicons, if available. When validation amplicons were not available, the amplicon used in the original library was used for validation. In some instances, both validation and library amplicons were used in validation experiments. In these cases, the knock-down result from the validation amplicon was preferred over that of the library amplicon, if a difference was evident. If the gene of interest was annotated to a pathway that had already been determined to be involved in MMS response, the gene was considered as a hit regardless of which version amplicon validated. Gene ontology Gene ontology was retrieved from FlyBase (http://flybase.org/). Enrichment analysis was conducted using FuncAssociate (http:// llama.med.harvard.edu/cgi/func/funcassociate/) for those ontol- ogies over-represented with a p-value of less than 0.05. Those ontology categories that overlapped were consolidated. Westerns Protein lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer containing 5% sodium deoxycholate, 0.1% SDS, 0.1% Igepal in PBS with a cocktail of protease inhibitors (1 mM PMSF, 1 mM Sodium Orthovandate and 30 uL/mL Aprotinin (Sigma)). Protein concentration was determined using Bradford Protein Assay Reagent (Biorad, Hercules, CA). Equal amounts of protein were resolved using 10% SDS polyacrylamide gel and transferred onto a nitrocellulose membrane (Hybond- ECL, GE Healthcare Lifesciences, Piscataway, NJ). Using appropriate dilutions of primary and secondary antibodies, immunodetection of the protein was performed using the ECL plus system (GE Healthcare Lifesciences). Primary antibodies include P-Chk1 (T68) (Abcam) and total CHK1 (Abcam), P- p70S6K (T389) (Cell Signaling Technology) and total p70S6K (Cell Signaling Technology), and P-p53 (S15) (Cell Signaling Technology) and total p53 (Cell Signaling Technology). Apurinic sites measurement Apurinic or apyrmidinic sites (AP) that were generated following DNA damage were measured using a colorimetric assay kit for DNA damage quantification (Oxford Biomedical Science, Oxford, MI), following manufacturer’s protocol. Briefly, DNA was isolated from the experimental cells using a Genomic DNA isolation kit (Oxford Biomedical Science). Equal amounts of DNA were then labelled using a biotinylated aldehyde reactive probe. Labelling was followed by purification and colorimetric quantification using streptavidin-horse radish peroxidase (HRP) conjugate and HRP- dependent substrate supplied with the kit. The aldehyde reactive probe labelled DNA standard, supplied by the kit, was used to determine the number of AP sites per 100 kilobase pairs of DNA in the experimental samples. Glutathione measurement Intracellular glutathione levels were measured using a colori- metric assay kit for glutathione (Oxford Biomedical Science), following manufacturer’s protocol. Briefly, cells from RNAi experiments were resuspended in ice-cold PBS and homogenized using a sonicator; the lysate was cleared by centrifugation and the protein concentration of the supernatant was determined using Damage Survival Pathways Bradford’s reagent (Biorad). For estimation of glutathione, equal amounts of protein lysate were de-proteinized using metapho- sphoric acid (MPA) with the final concentration of MPA adjusted to 5%. The precipitated proteins were cleared by centrifugation at 4000 g for 10 min at 4uC, and the supernatant was used for the assay. Reduced glutathione (Sigma) was used as a standard, and samples were arrayed in a 96-well plate. A two-step reaction was conducted, thioesterification of intracellular thiols using 4-choloro- 1-methyl-7-trifluoromethyl-quinolinium methylsulfate followed by alkaline conversion of glutathione-thioester to chromophoric thione, followed by detection of total glutathione by absorbtion at 400 nm. Quantitative Reverse-Transcriptase PCR dsRNA was used to target a gene of interest; this dsRNA was the same used to validate the genes of interest by knock-down. The level of RNAi mediated silencing of gene expression was monitored by quantitative real time RT–PCR using QuantiTect SYBR Green RT-PCR kit (Qiagen GmbH) and an ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, CA). For these experiments, 18.616104 Kc167 cells were dispensed into a 24- well tissue culture plate containing 6.2 mg of dsRNA per well. Following 1 h incubation in a serum-free condition to allow the uptake of dsRNA, serum was replenished to a final concentration of 10%. On Day 3, RNA was isolated using RNeasy Mini kit (Qiagen) and quantified using an ND-1000 Spectrophotometer (Nanodrop, Wilmington, DE). For the PCR amplifications, distinct primers that were not encompassed within the dsRNA used to target the gene were used. For gene expression analysis following MMS exposure, RNA was isolated on Day 4. For experiments with Kc167 cells, CG6905, the expression of which remained unaltered following MMS exposure, was used as an endogenous control. For experiments with MEFs, the isolated RNA was reverse transcribed using an ImProm-II Reverse Transcription System (Promega) and the cDNA was used with TaqMan Gene Expression Assay kit for GCLc, purchased from (Applied Biosystems, CA) and TaqMan Universal Master Mix (Applied Biosystems). Mouse b-Actin TaqMan Gene Expression Assay kit was used as an endogenous control. The level of gene expression was determined using nnCt method [50]. Proteasome measurement Experiments were performed in 384-well plates using the same layout and timing as described for the RNAi validation experiments, [18], except the proteasome activity was measured on Day 4 of the experiment, 24 h after MMS exposure. For proteasome measurement, a Proteasome-Glo assay kit was used (Promega), as per instructions. To normalize the proteasome activity with cell density, a parallel experiment was performed in a separate 384-well plate, and the cell density was estimated using CellTiter-Glo (Promega) as described above. Proteasome activity was then normalized to the relative number of cells present. Orthologue identification Orthologues of Drosophila genes of interest in human, mouse, and yeast, were obtained from Ensembl49 (http://ensembl.org/). siRNA targeting of MEFs SMARTPool siRNAs were purchased from Dharmacon RNA technologies (Lafayette, CO). For siRNA transfection into MEFs, cells were harvested by trypsinization, washed in a serum free medium, and cell density was adjusted to 16106 cells in 100 mL of MEF-2 Nucleofector Solution (Amaxa, Gaithersburg, MD), PLoS Genetics | www.plosgenetics.org 13 June 2009 | Volume 5 | Issue 6 | e1000527