is similarly altered in three or more other mouse aging profiles are indicated by an asterisk symbol. (PDF) Figure S11 Gene ontology biological process terms associated with mouse tail skin aging (CB6F1 strain). Genes regulated by aging in mouse tail skin were analyzed to identify significantly over-represented gene ontology (GO) biolog- ical process terms (P,0.05; Fisher’s exact test). The analysis was performed based upon (A) a set of 2633 mouse genes significantly increased by aging in females (P,0.05), (B) a set of 3134 mouse genes significantly decreased by aging in females (P,0.05), (C) a set of 2509 mouse genes significantly increased by aging in males (P,0.05) and (D) a set of 3208 mouse genes significantly decreased by aging in males (P,0.05). The font color of each GO term in the left margin corresponds to the degree to which that term was overrepresented (P,0.05, black font; P,0.01, blue font; P,0.001, red font). For each GO term, colors within the chart denote the average 2-year fold change (old/young) among genes associated with that GO term. The number of genes associated with each GO term is indicated in brackets (this total only includes genes that are among the 2633, 3134, 2509 or 3208 genes analyzed in parts A, B, C and D, respectively). Filled triangles indicate whether average fold-change estimates among these genes are significantly high (up-triangle; P,0.05) or significantly low (down-triangle; P,0.05). Triangles are displayed only if significant p-values were obtained from each of three separate tests evaluating whether the average fold-change estimate (or distribution of estimates) is significantly high or low (i.e., one-sample t-test, exact binomial proportion test and Wilcoxon signed rank test; triangles denote P,0.05 in each test). GO terms have been clustered based upon a similarity metric that is proportional to the number of shared ancestral terms in the GO hierarchy. (PDF) Figure S12 Lyz1 expression is not elevated with age in tail skin but increased by age in ear skin, heart and kidney from CB6F1 mice. RT-PCR was used to evaluate the expression of Lyz1 in mice from three age groups (5 months, 17 months and 30 months) and from both sexes (n = 5–6 mice for each sex/age combination). The expression of 18S ribosomal RNA (Rn18s) was used as an internal control gene for calculation of relative expression in each sample. Plots (A)–(F) show mean expression levels in each group (6 one standard error) normalized to the average expression level of the young (5 month) female group. In each plot, and for each sex, the average expression of middle-aged (17 month) and old groups (30 months) was compared to that of the young group (5 months). A red star indicates a significant difference relative to the youngest group of the same sex (P,0.05; two-sided two-sample t-test). A blue star is used to indicate a significant difference based upon a two-sample non-parametric statistical test (P,0.05; either Wilcoxon or Kruskal-Wallis rank sum test). (TIF) Figure S13 Lyz2 expression is not elevated with age in tail skin but increased by age in ear skin, heart, kidney and small intestine from CB6F1 mice. RT-PCR was used to evaluate the expression of Lyz2 in mice from three age groups (5 months, 17 months and 30 months) and from both sexes (n = 5–6 mice for each sex/age combination). The expression of 18S ribosomal RNA (Rn18s) was used as an internal control gene for calculation of relative expression in each sample. Plots (A)–(F) show mean expression levels in each group (6 one standard error) normalized to the average expression level of the young (5 month) female group. In each plot, and for each sex, the average Mechanisms of Transcriptome Aging expression of middle-aged (17 month) and old groups (30 months) was compared to that of the young group (5 months). A red star indicates a significant difference relative to the youngest group of the same sex (P,0.05; two-sided two-sample t-test). A blue star is used to indicate a significant difference based upon a two-sample non-parametric statistical test (P,0.05; either Wilcoxon or Kruskal-Wallis rank sum test). (TIF) Figure S14 Aging of mouse tail skin leads to decreased expression of transcripts associated with dendritic cells but increased expression of CD4+ T cell-associated genes (CB6F1 strain). We identified 500 probe sets (from the Affymetrix Mouse Genome 430 2.0 Array) associated with signature transcripts that exhibit high expression in CD4+ T-cells isolated from spleen (based upon data provided under GEO series accession GSE20366). Likewise, we identified 500 probe sets associated with transcripts that exhibit high expression in dendritic cells harvested from lung tissue (GSE18607). The scatterplots (A)– (D) display expression of these transcripts in tail skin samples from young (5 month) and old (30 month) CB6F1 mice of both sexes (n = 5 per age group for each sex). In each scatterplot, red symbols represent probe sets with higher expression in old mice, while green symbols represent probe sets with higher expression in young mice. The relative proportion of these two probe set groups is indicated by the pie chart shown in each panel. (TIF) Figure S15 Top-ranked motifs associated with aging effects in mouse tail skin (CB6F1 strain): Overlap with top-ranked human motifs but dissimilar associations with aging. This figure is identical to Figure 8, except a simulation-based correction for multiple testing has been applied (yellow symbols). For each simulation trial, genes were randomly assigned to gene sets (625 sets in total), where the respective size of gene sets was matched to those evaluated in our analysis. Following this random assignment of genes to gene sets, p-values were generated from each set to test for age-biased expression (Fisher’s Exact Test), and the lowest p-value arising among all gene sets was identified. This procedure was repeated in 2000 simulations, yielding a distribution for the minimum p-value expected to arise by chance alone, given the total number of gene sets evaluated for age-biased expression. We then identified the 0.05 quantile of this distribution (P*), which corresponds to the p- value that is lower than the minimal p-value that arose in 95% of the simulation trials. Those gene sets associated with p-values less than P* are denoted by yellow symbols. (TIF) Figure S16 Zeb1 and Prrx2 motif density near the TSS is associated with age-related expression patterns in tail skin from both sexes (CB6F1 strain). In each figure, blue or black symbols represent the average 2-year fold-change (old/ young) among genes with at least t binding sites in the region proximal to the annotated transcription start site (2 KB upstream and 200 BP downstream of the TSS). An increasing series of thresholds (t) was used to define gene sets of successively smaller sizes (horizontal axis), and for each set, the average 2-year fold- change was calculated (vertical axis). The background color (red or green) reflects the proportion of age-increased (red) to age- decreased genes (green) with respect to a given threshold (t), where an increase in the size of the red region denotes a higher proportion of age-increased genes, and an increase in the size of the green region denotes a higher proportion of age-decreased genes. For any given threshold t, blue symbols denote gene sets for which the average 2-year fold-change among genes with at least t PLoS ONE | www.plosone.org 18 March 2012 | Volume 7 | Issue 3 | e33204