decreased genes in females (green lines), (C, G, K) sequences within or adjacent to age-increased genes (red lines) in males, and (D, H, L) sequences within or adjacent to age-decreased genes in males (green lines). In each case, red or green lines represent the average residual score among foreground sequences (middle line) along with 95% confidence interval bounds (lower and upper lines). The grey region outlines a 95% confidence interval for the residual scores among sequences in the background set. Significant differences between the average foreground and background residual score are noted by blue bars near the top or bottom of each figure. Analyses were repeated using foreground sequence sets of varying size and selectivity (see horizontal axis). The smallest and most selective foreground set (leftmost-side of each figure) included only sequences adjacent to genes strongly altered by aging in the appropriate direction (based upon ranked p-values obtained from tests for an aging effect). The largest and least selective foreground set (rightmost-side of each figure) included sequences that were altered by aging in the appropriate direction, but were not necessarily adjacent to genes strongly altered by aging (based upon ranked p-values obtained from tests for an aging effect). (PDF) Figure S7 Weak association between aging effects in sun-protected human skin and tail skin from CB6F1 mice (adjusted residuals). A set of 15,203 human-mouse orthologous transcript pairs were placed into one of nine categories (horizontal axis in each panel) depending on whether transcripts were increased by aging (P,0.05; red up-triangles), decreased by aging (P,0.05; green down-triangles) or not altered by aging (black dash) in human sun-protected skin and/or tail skin from CB6F1 mice. For each category, an adjusted residual was calculated (vertical axis), which reflects departure from random association between human and mouse differential expression signatures (see Swindell [11]). Positive residuals indicate an overabundance of human-mouse transcript pairs within a particular category, while negative residuals indicate under- abundance of human-mouse transcript pairs within a category. Under the hypothesis (random association between human and mouse differential expression results), adjusted residuals are expected to follow a standard normal distribution, with residuals larger than three in absolute value providing evidence for a significant association between species. Correspondence between human-mouse aging patterns would have been supported by positive residuals for transcript pairs increased or decreased in both species (i.e., the fourth and third categories from the right), or alternatively, by negative residuals for transcript pairs with conflicting aging effects in the two species (i.e., the last two categories on the right). (TIF) Figure S8 Weak association between aging effects in sun-protected human skin and tail skin from CB6F1 mice (AUC statistics). 15,203 mouse transcripts were ranked according to the strength of aging effects among orthologous human genes in sun-protected skin (horizontal axis). This ranking was based upon (A) the degree of increase with aging in human females, (B) the degree of decrease with aging in human females, (C) the degree of increase with aging in human males or (D) the degree of decrease with aging in human males. In each panel (A)– (D), mouse transcripts associated with the strongest aging effect in humans (with respect to the human orthologue) were assigned lower ranks. Foreground sets of 200 mouse transcripts were then identified as those most strongly (A) increased with aging in mouse females, (B) decreased with aging in mouse females, (C) increased Mechanisms of Transcriptome Aging with aging in mouse males and (D) decreased with aging in mouse males. Red or green curves track the cumulative overlap (vertical axis) between the 200 foreground transcripts and the ranked set of 15,203 mouse transcripts. Enrichment of foreground genes with respect to the ranked lists (horizontal axis) is supported by red or green curves above the diagonal line, with a large positive area between the colored line and the diagonal (i.e., the region shaded in grey). This area is proportional to the Wilcoxon-Mann-Whitney (WMW) statistic, and for a large set of foreground genes chosen at random, is expected to follow a standard normal distribution (see equations 5–7 from Philippakis et al. [35]). The standard normal was therefore used as a distribution to evaluate the significance of WMW statistics obtained in (A)–(D) and to generate the p-value listed in the lower-right of each panel. (TIF) Figure S9 Weak association between aging effects in sun-protected human skin and tail skin from CB6F1 mice (Gene Set Enrichment Analysis). 15,203 mouse transcripts were ranked according to the strength of aging effects among orthologous human genes in sun-protected skin (horizontal axis). This ranking was based upon (A) the degree of increase with aging in human females, (B) the degree of decrease with aging in human females, (C) the degree of increase with aging in human males or (D) the degree of decrease with aging in human males. In each panel (A)–(D), mouse transcripts associated with the strongest aging effect in humans (with respect to the human orthologue) were assigned lower ranks. Foreground sets of 200 mouse transcripts were then identified as those most strongly (A) increased with aging in mouse females, (B) decreased with aging in mouse females, (C) increased with aging in mouse males and (D) decreased with aging in mouse males. The enrichment (vertical axis) is a measure of the cumulative overlap between the foreground set of mouse transcripts and the ranked list of 15,203 mouse transcripts (see Subramanian et al. [36]). The enrichment score (ES) metric is the maximum enrichment observed across all ranked genes (see red circle) and is used as the test statistic. The distribution of ES was generated from 1000 simulation trials in which the 15,203 mouse transcripts were ranked based upon randomized expression data. The shaded grey region within each panel represents the lower 95% of the simulated distribution for ES. Correspondence between human-mouse aging patterns is not supported in (A)–(D) because the red circle (ES) shown in each panel lies within the grey region (i.e., within the lower 95% of the ES distribution). (TIF) Figure S10 Ranked lists of genes altered by aging in mouse tail skin and other tissues. Tables display age- associated expression patterns for ranked lists of the top 50 genes (A) increased by aging across all mouse tissues, (B) decreased by aging across all mouse tissues, (C) increased by aging in mouse tail skin (CB6F1 strain), or (D) decreased by aging in mouse tail skin (CB6F1 strain). For a given gene (row) and tissue (column), colors denote the estimated fold-change expression ratio between an older mouse t+2 years of age and a younger mouse t years of age (a linear rate of change with age is assumed). Filled triangles denote significant aging effects (P,0.05). In (A) and (B), gene rankings are based upon the total number of significant results observed across all columns in each table, and an asterisk symbol is used to denote genes similarly altered between mouse skin (males and/or females) and the other aging profiles. In (C) and (D), genes were first filtered to include only those significantly altered by aging in skin (P,0.05), and then ranked according to fold-change estimates (old/young) (averaged between sexes). Genes for which expression PLoS ONE | www.plosone.org 17 March 2012 | Volume 7 | Issue 3 | e33204