Damage Survival Pathways Figure 4. An examination of the functionality of five of pathways utilized in MMS survival by Drosophila cells. (A) MMS exposure results in an increase in AP sites. dsRNA knock-down of the BER component XRCC1 increases the quantity of AP sites observed following MMS exposure. (B) MMS exposure results in increased p53 expression by quantitative real-time PCR analysis; expression level is provided as fold change compared to an endogenous control (CG6905). (C) 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). The intracellular glutathione concentration is expressed as mmoles/L normalized per mg protein. This GCLc dependency correlates with the increased level of GCLc expression observed following MMS exposure. (D) Proteasome activity is increased following MMS exposure in a manner that is dependent upon proteasome components Rpn2 and Pros26.4. Proteasome activity is expressed as units of activity/cell, normalizing the activity to the number of cells using a parallel viability assessment. (E) MMS exposure results in a dose-dependent decrease in the phosphorylation of the dTor component p70S6K. This decrease is dependent upon the negative regulator of dTor, Tsc1. Statistically significant differences between unexposed and MMS exposed cells are denoted (*; **; ***; with p-values of ,0.05; ,0.01; and ,0.001 respectively) as are differences between Luc control and each dsRNA knock-down (#; ##; ###; with p-values of ,0.05; ,0.01; and ,0.001 respectively). doi:10.1371/journal.pgen.1000527.g004 Figure 5. Functional conservation in mouse embryonic fibroblasts of five pathways found to be utilized for MMS survival in Drosophila cells. (A) MMS exposure results in an increase in AP sites, siRNA based XRCC1 knock-down results in an increased quantity of AP sites following MMS exposure. (B) MMS exposure results in phosphorylation of CHK1 and p53 and in an accumulation of total p53 levels. (C) MMS exposure results in an increased amount of total glutathione and this increase is partially dependent upon GCLc. The intracellular glutathione concentration is expressed as mmoles/L normalized per mg protein. This GCLc dependency correlates with the increased level of GCLc expression observed following MMS exposure. (D) Proteasome activity is increased following MMS exposure in a manner that is dependent upon proteasome components Pmsc1 and Psmd1. Proteasome activity is expressed as units of activity/cell, normalizing the activity to the number of cells using a parallel viability assessment. (E) MMS exposure results in a dose-dependent decrease in the phosphorylation of p70S6K. Statistically significant differences between unexposed and MMS exposed cells are denoted (*; **; ***; with p-values of ,0.05; ,0.01; and ,0.001 respectively) as are differences between NT control and each siRNA knock-down (#; ##; ###; with p-values of ,0.05; ,0.01; and ,0.001 respectively). doi:10.1371/journal.pgen.1000527.g005 PLoS Genetics | www.plosgenetics.org 9 June 2009 | Volume 5 | Issue 6 | e1000527