Correction: Mice Fed Rapamycin Have an Increase in Lifespan Associated with Major Changes in the Liver Transcriptome

Figure S1: Body weight data for mice from 5 to 21 months of age. Body weight data for control and rapamycin (Rapa) male (A) and control and Rapa female (B) mice are shown from 5 to 21 months of age for 15 mice in each group. The data are the mean ± standard error of the mean (SEM) statistically analyzed by Two-way ANOVA with repeated measure followed by Tukey’s pairwise comparisons. The data that were statistically significant with p<0.05 are indicated by *. * * * * * * * * * * * * * * * * * A.

: Body weight data for mice from 5 to 21 months of age. Body weight data for control and rapamycin (Rapa) male (A) and control and Rapa female (B) mice are shown from 5 to 21 months of age for 15 mice in each group. The data are the mean ± standard error of the mean (SEM) statistically analyzed by Two-way ANOVA with repeated measure followed by Tukey's pairwise comparisons. The data that were statistically significant with p<0.05 are indicated by *. B. Figure S2: Food consumption data for mice from 5 to 24 months of age. Food consumption data is presented as average consumption per week per mouse for control and Rapa male (A) and control and Rapa female (B) mice from 5 to 24 months of age. Food consumption was measured per week minus the food not consumed at the bottom of the cage and calculated per mice in the cage. Food consumption was measured for 3 cages per group with 5 mice in each cage. The data are the mean ± SEM and were statistically analyzed by Two-way ANOVA with repeated measure follow by Tukey's pairwise comparisons. We did not observe any statistical difference between Rapa and control for both males and females.   Table S2: Fitted Gompertz Mortality Models Ln plots of fitted Gompertz mortality models where the hazard (instantaneous mortality rate) at age x, u x = ae bx , where x begins at 4 months and continues until the end of life. The parameters for the Gompertz model are shown with Gompertz parameter a as the initial mortality rate at 4 months and, Gompertz b as the rate of increasing mortality. In the parenthesis are the 95% confidence interval for parameters Gompertz a and b. Statistical analysis was done using maximum-likelihood estimation and generating chi-square tests for comparison of constrained and freely varying models. The p-values indicating the level of confidence in equating model parameters are given. We found that the whole mortality model differed between Rapa and Control in both females (p<0.0001) and males (p=0.0039). Pooling of the sexes allowed for identifying the parameter(s) that differ between Rapa and control-fed animals, and indicates that the rate of aging (b), but not the initial mortality rate (a), differed between Rapa and control mice.

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D. Figure S4: Gene analysis shows that Rapa-2 males share many genes that change in Rapa Female mice.
We identified the transcripts that were significantly changed by Rapa using a false discovery rate of adjusted p-value <0.05 and >15% change relative to respective controls for each sex. As described in Figure 2, the male mice fed Rapa appeared in analysis to segregate as 2 groups, which will be shown as Rapa-1 and Rapa-2. The venn diagrams show the number of transcripts that were significantly up-regulated (A) or down-regulated (B), in Rapa-1 male and Rapa female mice, and the transcripts that were significantly up-regulated (C) and downregulated (D) in Rapa-2 male and Rapa female mice. The Rapa male mice are represented in pink, Rapa female mice in cyan, and the yellow indicates sharing of Rapa male and female mice.

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C. B.

Figure S5: Validation of genes in the mitochondria dysfunction and estrogen receptor signaling pathways.
Using qRT-PCR, we measured the levels selected transcripts that were observed to change in the mitochondria dysfunction (A) and estrogen receptor signaling (B, C) pathways. The data shown are the means relative to respective sex controls ± SEM. The data were analyzed using student t-test, and the p-values are shown. For the colors, Control male is in red, Rapa-2 male in pink, control female in blue and Rapa female in cyan. A list of the primers used in the qRT-PCR validation is listed (D).   We identified the transcripts that were significantly changed by 6 months of Rapa treatment using a false discovery rate of adjusted p-value <0.05 and >15% change relative to respective controls for each sex. The venn diagrams show the number of transcripts that were significantly up-regulated (A) or down-regulated (B), in 6-months Rapa male and Rapa female mice. The Rapa male mice are represented in pink, Rapa female mice in cyan, and the yellow indicates sharing of Rapa male and female mice. Pathway analysis was conducted using ingenuity pathway analysis for genes that were significantly changed in 25month-old females fed Rapa for 6 months relative to control females with adjusted p<0.05 and >15% change (C). The pathway analysis used Fisher's exact test to place genes into pathways and we also used a FDR cutoff of B-H p<0.05 to show the pathways that were highly significant. All the pathways (3) with a B-H p<0.05 are shown. Two of the three pathways, protein ubiquitination and mitochondrial dysfunction, are also changed in chronic Rapa female. For the graphical portion, the yellow line indicates the -log(B-H p-value). Red indicates up-regulated and green for down-regulated genes in our transcriptome analysis found to be significantly changed in Rapa compared to control, and white indicates the percentage of genes not significantly changed in the pathway relative to the total number of genes in that pathway (bolded number). A list of the pathways that significantly changed with 6-months Rapa feeding is shown in in File S2, tab5 for Rapa female and a list of the transcripts that change in protein ubiquitination and mitochondrial dysfunction is shown in File S3 tab14 and tab15 for 6-months Rapa female compared to chronic Rapa female. Mouse primary dermal fibroblasts from 3-6 month old C57BL/6 mice at passage 2 were pretreated with 0 (white bars), 100 (black bars) or 300 (grey bars) nM of Rapa for 24 hours. Cells were then washed and the media was then replaced. Cells were treated with hypochlorous acid at 0.04% and 0.05% (A), paraquat at 2.5mM and 5 mM (B), and tert-Butyl hydroperoxide at 50μM and 100μM (C) for 6 hours after which cells were washed and media was replaced. After 18 hours, cell viability was determined using a microplate reader. The data are the mean ± SEM are shown for 2 independent of replicates. The asterisk indicates p<0.05 to 0 nM Rapa and the number sign indicates p<0.05 to 100 nM. Addition of Rapa significantly decreases cell survival with all the stressors.
File S2: File (File S2.xlsx) containing data for genes and pathways found to be significantly changed by Rapa. Tab1 ("1. Genes-Chronic Rapa"): A list of all genes found to be significantly different in chronic Rapa male or female (using criteria of adjusted p<0.05) from respective sex controls. The list is sorted in the order of the gene hierarchical cluster of the heatmap in Figure 3. The group column indicates where the genes are found in the Venn diagram ( Figure S4A-S4D). The log change is colored with red for significantly up-regulated genes, green for significantly down-regulated genes, and white for non-significant genes. Tab2 ("2. IPA Rapa-2 Male|Control Male"): IPA pathways for genes significantly altered in chronic Rapa-2 male/control male with a B-H p<0.05, ranked from lowest to the highest B-H p-value. Tab3 ("3. IPA Rapa Female|Control F"): IPA pathways for genes significantly altered in Rapa female/control female with a B-H p<0.05, ranked from lowest to the highest B-H pvalue. Tab4 ("4. Genes-6-months Rapa"): A list of all genes found to be significantly different in 6-months Rapa male or female (using criteria of adjusted p<0.05) from respective sex controls. The list is sorted in the order of the gene hierarchical cluster of the heatmap in Figure 6B. The group column indicates where the genes are found in the Venn diagram ( Figure S6A and S6B). The log change is colored with red for significantly up-regulated genes, green for significantly down-regulated genes, and white for non-significant genes. Tab5 ("5. IPA 6-months Rapa F|Ctrl F"): IPA pathways for genes significantly altered in 6months Rapa female/control female with a B-H p<0.05, ranked from lowest to the highest B-H p-value. Tab6 ("6. Genes-6-mon vs. Chronic Rapa"): A list of all genes found to be significantly different in 6-months Rapa female or chronic Rapa female (using criteria of adjusted p<0.05) from respective controls and matched from both microarray sets. The list is sorted in the order of the gene hierarchical cluster of the heatmap in Figure 7A. The group column indicates which group the gene is found significant in. The log change is colored with red for significantly up-regulated genes, green for significantly down-regulated genes, and white for non-significant genes.
Tab14 ("14. Mito Function-RF6 vs RFL"): A list of all genes for mitochondria dysfunction pathway found to be significantly changed in 6-months Rapa female or chronic Rapa female compare to respective controls. Red indicates up-regulated genes, green indicates downregulated genes, and white indicates no change. Tab15 ("15. Protein Ubiq. RF6 vs RFL"): A list of all genes for protein ubiquitination pathway found to be significantly changed in 6-months Rapa female or chronic Rapa female compare to respective controls. Red indicates up-regulated genes, green indicates downregulated genes, and white indicates no change.