analysis and convincing evidence that the pathways were indeed functioning as expected, we were not able to assign every protein within each pathway as an ‘‘MMS survival protein.’’ There are many possible explanations for this, but nonetheless, considering the pathways as a whole provide a framework within the network that highlights novel interactions, cross-talk, and identified proteins not mapped to a canonical pathway, but present within the network, would unlikely be observed in a ‘‘chaotic network view’’ and encourages their investigation. This approach is similar to computational clustering of networks based on signaling pathways using interactome datasets [35], but our approach includes both identified proteins and pathway components. Our representation is simplified, using a single node to represent the entire pathway rather than a complex display of interactions for every component in a pathway [36]. This simplified pathway inclusive representation reveals a highly organized network, consistent with the requirement of each pathway for cellular survival (Figure 6E), and provides an effective strategy to integrate modular components into the network [37] and thereby inferring biological properties [38]. The interconnectivity between the survival pathways (Figure 5E) suggests potential pathway cross-talk. If such cross- talk exists, it would be highly pertinent to cancer therapy. Recent studies have demonstrated the utility of a global level analysis, allowing identification of altered pathway function in complex diseases such as the Notch pathway in pancreatic cancer [39] and similarly the DDR pathway in breast and colorectal cancers [40]. Considering our identification of Notch, TOR, DDR, and the proteasome as ‘‘survival pathways,’’ all of which are currently being explored as targets for cancer therapy [41,42], our identified survival network would suggest the possibility of combining pathway-specific pharmacological agents in cancer therapy. Some of the pathway connections and potential cross-talk represented in our survival network (Figure 6E) have already been observed. For example, protein phosphatase, PP2A, a downstream component of the TOR pathway [23], interacts with the DDR component to regulate phosphorylation of ATM and ATR [43] and vice versa [4]; DDR interacts with BER via CHK2 and XRCC1 [44]; Notch interacts with DDR via Mastermind and p53 [45]; the glutathione pathway interacts with the nucleotide excision repair pathway (NER) [46]; and the proteasome interacts with various DNA repair components [34,47]. Together, it would appear that our model of an integrated network of conserved damage survival pathways is both valid and biologically relevant. In conclusion, we have identified a network of pathways that have a functional role in damage response by affecting viability; we also demonstrated the functional conservation between species of the MMS survival pathways. By considering the protein interactions between the MMS survival proteins and by incorpo- rating the MMS survival pathways, a highly interconnected damage survival network is observed that encompasses at least 58% of the identified MMS survival proteins directly. This interconnectivity suggests a strong functional interrelationship between constitutive components of the survival network and the possibility of pathway cross-talk and coordination at a level greater than just their instigation by the DDR pathway. Although these MMS survival pathways have already been implicated in MMS damage response, we have identified these seemingly disparate pathways in a single screen, and with the network analysis, this lends to direct connection of these pathways in response to damage. The pleiotropic effects of alkylation damage therefore require a wide-variety of functioning pathways in order for the cell to survive. Damage Survival Pathways Methods Tissue culture Kc167 cells were grown in Schneider medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated fetal bovine serum (65uC, 10 minutes), penicillin and streptomycin at 22uC in a humidified chamber. Primary MEFs were obtained by harvesting 14.5 day C57BL/6 embryos as previously described [48]. Briefly, fetal liver and head were removed and the remainder of the embryo mechanically disaggregated in plating medium. A suspension of single-cells was plated out in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. MEFs were grown for two passages before freezing aliquots. Aliquots were taken and expanded as needed for each experiment. Genome-wide viability screen following MMS exposure Methods were adapted from [49], with 1.26104 Kc167 cells suspended in 10 mL of serum-free Schneider medium (Invitrogen) added to each well of sixty-three 384-well plates. Each well contains about 0.25 mg of a double-stranded (ds) RNA, with 22,915 individual dsRNA represented in the whole library (Version 1 dsRNA). Cells were incubated for 1 h at 22uC, allowed phagocytic uptake of the dsRNA, then 20 mL of serum (15% heat- inactivated fetal bovine serum) containing medium were added and the plates incubated for a further 72 h. Medium was exchanged for fresh serum containing medium, with or without 0.004% (w/v) of MMS (Sigma-Aldrich, St. Louis, MO). Following 72 h additional incubation, medium was removed and the number of viable cells assessed using pre-diluted Celltiter-Glo per manufacturers instructions (Promega, Madison, WI). Screens were performed in duplicate, with the replicate screen initiated on a different day. Production of dsRNA dsRNA were produced similarly to previously described [8,18]. Briefly, dsRNA was synthesized using cDNA prepared by gene specific amplification of reverse transcribed cDNA. For cDNA preparation, RNA was harvested from adult D. melanogaster using Trizol reagent. For gene specific amplification, primers designed by the DRSC to target genes of interest were custom synthesized with overhanging T7 promoter sequences (Invitrogen). DRSC validation primer sequences were obtained from the DRSC for targeting genes of interest, and oligonucleotides were purchased (Invitrogen). First round PCR amplifications were performed using gene specific primers, and the amplified products were gel purified, using an agarose gel purification kit (Qiagen, Valencia, CA) or a 96-well gel isolation kit (Invitrogen). The purified PCR products were then used for second round amplification also using the gene specific primers; these amplified products were purified using Millipore PCR purification 96-well plate system (Millipore, Billercia, MA). The purified products were then used for dsRNA synthesis using T7 Ribomax express system (Promega) and purified using the PCR purification plate system (Millipore). The quality of purified dsRNA was verified by agarose gel electropho- resis and, following spectrophotometric quantification, stored at 220uC. Validation analysis Validation of MMS survival genes was performed as previously described [18], with the exception that DRSC ‘‘validation’’ dsRNA (targeting a gene mRNA transcript at a different location to the library amplicon), designed by the DRSC but produced PLoS Genetics | www.plosgenetics.org 12 June 2009 | Volume 5 | Issue 6 | e1000527