microscopic examination with 8-oxoguanine containing cells observed by fluorescence in the FITC channel and this (B) can be quantified as a percentage of fluorescent within a field (ordinate) for increasing dose of MMS (abscissa). Found at: doi:10.1371/journal.pgen.1000527.s007 (7.71 MB EPS) Figure S8 Temozolomide exposure results in functional re- sponse by MMS survival pathways in mouse embryonic fibroblasts. (A) Temozolomide exposure results in phosphorylation of p53 and in an accumulation of total p53 levels. (B) Temozolomide exposure results in an increased amount of total glutathione. The intracellular glutathione concentration is ex- pressed as units of activity/cell. (C) Proteasome activity is increased following temozolomide exposure, normalizing the activity to the number of cells using a parallel viability assessment. Proteasome activity is expressed as units of activity/cell, normalizing the activity to the number of cells using a parallel viability assessment. Found at: doi:10.1371/journal.pgen.1000527.s008 (0.71 MB EPS) Figure S9 MMS and Temozolomide exposure results in functional response by notch signalling pathway in HEK 293 cells. MMS and temozolomide exposure results in decreased luciferase activity of the notch reporter RBP-Jk, normalized for transfection efficiency in HEK 293 cells using renilla luciferase. Found at: doi:10.1371/journal.pgen.1000527.s009 (0.41 MB EPS) Figure S10 Validation of pathway functions with additional siRNA knock down in mouse embryonic fibroblasts. (A) Knock- down of GCLc with four different siRNA results in decreased GCLc expression in mouse cells, by quantitative real-time PCR analysis; expression level is provided as fold-change compared to an endogenous control (mouse b-actin). (B) MMS results in an increased amount of total glutathione, and this increase is dependent upon the rate limiting glutathione metabolizing enzyme glutamate-cysteine ligase (GCLc). (C) Knock-down of proteasome components Psmc1 or Psmd1 with four different siRNA results in decreased gene expression in mouse cells, by quantitative real-time PCR analysis; expression level is provided as fold-change compared to an endogenous control (mouse b-actin). (D) Proteasome activity is increased following MMS exposure in a manner that is dependent upon proteasome components Pmsc1 and Psmd1. Found at: doi:10.1371/journal.pgen.1000527.s010 (0.81 MB EPS) Table S1 Summary of MMS screen hits and pathway genes selected for validation. The 534 Drosophila MMS screen hits selected for validation analysis and 298 pathway genes tested for pathway analysis, provided with FlyBase gene number, corre- sponding CG number, and the dsRNA used for validation, noted by DRSC identification number, either library dsRNA or validation dsRNA. For those genes that had no validation amplicon designed or those whose library amplicon had no potential off-target effects at 19 nt, data from [18] was used (asterisks). For each dsRNA, significant death, trend death or significant resistance to MMS treatment is noted (MMS survival). Found at: doi:10.1371/journal.pgen.1000527.s011 (0.94 MB XLS) Table S2 Raw, normalized, and survival data for validation experiment. For data normalization, raw data of untreated control and MMS experiments were normalized using luciferase (Luc) and high MMS controls and statistical significance was determined as described [18]. Found at: doi:10.1371/journal.pgen.1000527.s012 (0.23 MB XLS) Damage Survival Pathways Table S3 List of yeast MMS hits and their fly orthologues. Yeast MMS hits as determined by Begley et al. [3], and their fly orthologues that were neither a hit in our MMS screen nor in a pathway identified from the screen. Also given is whether each fly gene was essential, a resistance hit, a death hit, or a death tread after validation with an independent dsRNA. Found at: doi:10.1371/journal.pgen.1000527.s013 (0.04 MB XLS) Table S4 Drosophila GST family members and their involvement in MMS survival. The five GST families and their component members. Provided is the gene name, corresponding CG number, whether they validated as a significant MMS survival gene (death), a trend (death trend) or not involved in MMS survival (no). For each validation observed to have an effect, the type of amplicon, library or validation is given. It is also noted for any validated MMS survival gene whether the effect was also observed in the screen. If validation was not performed this is also noted (not tested). Found at: doi:10.1371/journal.pgen.1000527.s014 (0.02 MB XLS) Table S5 Summary of the number of MMS survival genes identified per MMS survival pathway (BER: base excision repair; DDR: DNA damage response; Proteasome; GSH: glutathione synthesis; TOR: TOR pathway; NER: nucleotide excision repair; MMR: mismatch repair; HRR: homologous recombination repair; RECQ: RecQ helicases; Transcription: basal transcription; Ribosome; ATPase; Notch: Notch signaling pathway), and the percentage non-essential genes of each pathway that this represents. Protein enrichment within each pathway compared to the number of genes that validated (202 of 13826 prior to pathway analysis and 307 of 13826 including pathway analysis) is determined using a Fisher’s Exact Test (NS: Not Significant; NA: Not Applicable). Found at: doi:10.1371/journal.pgen.1000527.s015 (0.51 MB PDF) Table S6 The orthologous relationship between the MMS survival genes identified in the Drosophila and yeast MMS survival screens. Provided are the 13 MMS survival pathway, the gene names of each Drosophila component and their corresponding CG number. For each of these Drosophila genes, the yeast ORF is provided for any identifiable yeast orthologue and whether it was observed as being involved in MMS survival (hit) in S. cerevisiae by Begley et al. [3], if it was not identified (not hit) or if it is essential (essential). Where orthologues between the species are known and taking into account the yeast essential genes that could not be assayed for their effect on MMS survival, there is a clear enrichment in MMS survival genes within the majority of pathways examined between the two species. A complete list of these proteins and their human orthologues are also given. Found at: doi:10.1371/journal.pgen.1000527.s016 (0.11 MB XLS) Table S7 Connectivity analysis of MMS survival genes identified by the RNAi genomic screen and following validation analyses. Provided are the number of proteins identified, the number of those proteins that are within the protein:protein interactome (PPI), the number of direct connections between these proteins, the average distance between every possible pair of proteins within the network compared to the expected values, the global efficiency and the clustering coefficient. For each measurement the expected number of direct interactions is derived from the same number of proteins randomly selected 1000 times from the PPI. Analyses are provided for the proteins identified by the MMS screen prior to validation (Screen hits), those screen hits that actually validated PLoS Genetics | www.plosgenetics.org 16 June 2009 | Volume 5 | Issue 6 | e1000527