Supporting Information Figure S1 The MMS dose response as measured by cell survival. Viability of Drosophila Kc167 cells following exposure to increasing dose of MMS determined using CellTiter-Glo (relative light units (ordinate) for increasing dose of MMS (abscissa)). Found at: doi:10.1371/journal.pgen.1000527.s001 (0.54 MB EPS) Figure S2 Four of the eight additional pathways utilized for MMS survival. CG numbers are given for each Drosophila pathway component, as well as the protein names or complex names for their human orthologues. Pathway entry points are noted with Roman numerals at the top, and end points are at the bottom. A key for the following symbols is provided. Symbols encircled with thick lines represent proteins that act together or in a complex, while symbols encircled with thin lines represent paralogues or proteins that may substitute for one another. Proteins found to affect MMS survival are noted as down (death) or up triangles (resistance). Statistically significant proteins are indicated with black triangles, while trend hits are indicated with grey triangles. Essential genes are noted with a thick bar and any with downwards or upwards pointing boxes were also validated as conferring death or resistance, respectively, to MMS upon knock- down. Shaded squares are proteins not found to be hits after validation, and open squares were not tested in our validation. Yeast orthologues previously found to be required for MMS survival [3] are noted with a dot under the symbol. An example of average percent of untreated control survival of validated hits is shown next to each pathway, though this may not represent the actual control for each data point within the graph. Error bars are the standard deviation of quadruplicates. Survival of control cells with dsRNA targeting luciferase is shown in an open bar, protein knock-down that resulted in a significant difference in MMS survival from this control are shown in black (death) or stripped (resistance), and those with a trend effect are shown in grey. A complete list of these proteins and their human and yeast orthologues is given in Table S6. (A) Nucleotide Excision Repair. (B) Mismatch Repair. (C) Homologous Recombination Repair. (D) RecQ Helicases. Found at: doi:10.1371/journal.pgen.1000527.s002 (1.40 MB EPS) Figure S3 Two of the eight additional pathways utilized for MMS survival. CG numbers are given for each Drosophila pathway component, as well as the protein names or complex names for their human orthologues. Pathway entry points are noted with Roman numerals at the top, and end points are at the bottom. A key for the following symbols is provided. Symbols encircled with thick lines represent proteins that act together or in a complex, while symbols encircled with thin lines represent paralogues or proteins that may substitute for one another. Proteins found to affect MMS survival are noted as down (death) or up triangles (resistance). Statistically significant proteins are indicated with black triangles, while trend hits are indicated with grey triangles. Essential genes are noted with a thick bar and any with downwards or upwards pointing boxes were also validated as conferring death or resistance, respectively, to MMS upon knock- down. Shaded squares are proteins not found to be hits after validation, and open squares were not tested in our validation. Yeast orthologues previously found to be required for MMS survival [3] are noted with a dot under the symbol. An example of average percent of untreated control survival of validated hits is shown next to each pathway, though this may not represent the actual control for each data point within the graph. Error bars are the standard deviation of quadruplicates. Survival of control cells with dsRNA targeting luciferase is shown in an open bar, protein Damage Survival Pathways knock-down that resulted in a significant difference in MMS survival from this control are shown in black (death) or stripped (resistance), and those with a trend effect are shown in grey. A complete list of these proteins and their human and yeast orthologues is given in Table S6. (A) Basal Transcription. (B) Ribosome. Found at: doi:10.1371/journal.pgen.1000527.s003 (3.26 MB EPS) Figure S4 Two of the eight additional pathways utilized for MMS survival. CG numbers are given for each Drosophila pathway component, as well as the protein names or complex names for their human orthologues. Pathway entry points are noted with Roman numerals at the top, and end points are at the bottom. A key for the following symbols is provided. Symbols encircled with thick lines represent proteins that act together or in a complex, while symbols encircled with thin lines represent paralogues or proteins that may substitute for one another. Proteins found to affect MMS survival are noted as down (death) or up triangles (resistance). Statistically significant proteins are indicated with black triangles, while trend hits are indicated with grey triangles. Essential genes are noted with a thick bar and any with downwards or upwards pointing boxes were also validated as conferring death or resistance, respectively, to MMS upon knock- down. Shaded squares are proteins not found to be hits after validation, and open squares were not tested in our validation. Yeast orthologues previously found to be required for MMS survival [3] are noted with a dot under the symbol. An example of average percent of untreated control survival of validated hits is shown next to each pathway, though this may not represent the actual control for each data point within the graph. Error bars are the standard deviation of quadruplicates. Survival of control cells with dsRNA targeting luciferase is shown in an open bar, protein knock-down that resulted in a significant difference in MMS survival from this control are shown in black (death) or stripped (resistance), and those with a trend effect are shown in grey. A complete list of these proteins and their human and yeast orthologues is given in Table S6. (A) ATPases. (B) Notch. Found at: doi:10.1371/journal.pgen.1000527.s004 (2.47 MB EPS) Figure S5 Average percent of untreated control survival of validated hits in each pathway represented in Figures 2 and 3. Error bars are the standard deviation of quadruplicates. Survival of control cells with dsRNA targeting luciferase is shown in an open bar as an average across all plates as a general reference. It should be noted that each plate had its own luciferase controls against which all plate values were compared. Protein knock-down that resulted in a significant difference in MMS survival from their internal plate control are shown in black (death) or dashed (resistance), and those with a trend effect are shown in grey. A complete list of these proteins and their human and yeast orthologues is given in Table S6. Found at: doi:10.1371/journal.pgen.1000527.s005 (4.48 MB EPS) Figure S6 Efficiency of RNAi mediated silencing of gene expression. (A) RNAi transfection resulted in decreased expression of target mRNA in Drosophila Kc167 cells, measured by quantitative real time PCR as indicated percent control gene expression (ordinate) for the genes (abscissa) tested, with expression normalized to endogenous control (CG6905). (B) Western blot analysis for efficiency of RNAi in siRNA transfected primary mouse embryonic fibroblast cells. Found at: doi:10.1371/journal.pgen.1000527.s006 (0.74 MB EPS) Figure S7 MMS exposure results in a dose-dependent increase in 8-oxoguanine DNA modifications. The MMS-dependent increase in 8-oxoguanine is (A) observed qualitatively by PLoS Genetics | www.plosgenetics.org 15 June 2009 | Volume 5 | Issue 6 | e1000527