A B

Young mouse Old mouse skin skin

p65

p65 levels: 1.0 1.0 1.0 1.3 Human kidney Medulla Human kidney Cortex Human muscle (Arm/Leg) Human fibroblasts Human muscle (Abdomen) Human brain NFKBIA p105 CHUK NFKB1 IL1R1 PTPLAD1 MYD88 p50 NKIRAS1 IL1A p105 levels: 1.0 1.3 1.3 1.1 TRAF6 p50 levels: 1.0 1.1 1.6 1.2 TLR8 MALT1 MAP3K7 IκBα TNFRSF10A STAT1 IκBα levels: 1.0 1.1 0.8 0.9 CARD10 IRAK1 CARD14 ACTIN TNFRSF10B TNFRSF1A IKBKB MAP3K1 TIAF1 TNFAIP3 MAP3K7IP1 FADD B pathway genes (Biocarta)

κ NKIRAS2 TNF

NF- TLR1

ZNF675 TBK1 TLR6 MAP3K14 RIPK1 TRADD IKBKG TLR4 TLR3 RELA

Genes induced with age (P<0.05) Genes repressed with age (P<0.05)

Supplementary Figure 1. Diverse mechanisms of increased NF-κB activity with age. (A) Western analysis of two independent young and old mouse skin samples shows that NF-κB subunits p65, p50, and p105 weakly increase with age, while IκBα protein expression weakly decreases with age. Quantification was performed with ImageJ (http://rsb.info.nih.gov/ij/). (B) Expression of 38 genes found in NF-κB signaling pathways (BioCarta Molecular Pathways: http://www.biocarta.com) were anlayzed in six human tissues. One-sided t-tests identified genes whose expression significantly changes with age (P<0.05) in the indicated tissue. Some genes showed consistent change in multiple tissues, while many genes showed tissue specific effects. compared to GFP orNFKB1 motif combinationssimilarly affectedbyNFKB1 microarrays; averagedata ofduplicatesamplesareshown.Leftbottom:Analysis ofmotifmodulesidentifiesspecific (labeled withCy5;red)and control-treatedsamples(labeledwithCy3;green)were competitivelyhybridizedto I strongly inhibitsTNF- Right: InGFP-expressingcells,TNF- supershift assaywiththeindicatesantibodiesidentifies p65:p50asthepredominantheterodimerinkeratinocytes. interaction isdetectedwithRelB,cRel,orp52.(C)NFKB1 endogenous p65ina4-OHT-dependentfashion,weakly withp50inaligand-independentfashion,andnoappreciable (B) Immunoprecipitation(IP)ofNFKB1 NFKB1 Supplementary Figure2. C A κ B α

-SR (superrepressor).For keratinocytestransducedwitheachoftheindicated genes, TNF- ---- 4-OHT 2h 4-OHT 0h GFP +TNFAntibody ∆ SP α-p50 -ER (green)translocatestothenucleus(DAPI,blue) of keratinocytesuponaddition4-OHTfor2hours. α-p65

α-cRel + LacZ α

α-p52 NFKB1 -induced heterodimerformation. (D)NFKB1

α-RelB Characterization ofNFKB1 ∆ ∆ SP SP 4-OHT: -ER localization TNF: -ER (no4-OHT) arealsorepressedbyNFKB1 + - - NFKB1 α stronglyinducesheterodimerformation,whileexpression ofNFKB1 ∆ GFP SP + - -ER inkeratinocytesrevealsthatNFKB1 ∆ SP + + -ER ∆ NFKB1 SP + - -ER andI ER + + ∆ ∆ SP SP - -ER specificityinprimaryhumankeratinocytes.(A) ∆ SP κ D B -ER blocksp65:p50NF- B 4-OHT: α Average exp.targets -SR. Right:Genesthatare repressedbyI -1.5 ∆

SP GFP +TNF GFP ∆SP - -ER inhibitsthesameTNF- NFKB1 -ER IκBα-SR GFP +1.5 IκB-α-SR + ∆SP Input NFKB1∆SP-ER + 4-OHT NFKB1 -ER GFP NF- NF- NF- AP2- AP1 :ZID

- ∆SP ∆ NFKB1 I ACTIN NFKB1 -ER κ κ κ κ SP B B :GATA4 B :CCANNAGRKGGC B :IRF α + α :POU1F1RREB1 ∆ -ER, albeitto a lesserextent. SP

-SR NFKB1 -ER ∆ SP ∆ SP κ -ER interactsstronglywith -ER B heterodimer.Left:EMSA - GFP IP +

GFP α -ER

- NFKB1∆SP-ER α α +

-treated samples ∆SP -induced genesas NFKB1 -ER p52 cRel RelB p50 p65 NFKB1 GFP +TNF ∆SP κ NFKB1 -ER ∆ -2.5 ∆ SP B SP -ER α ∆SP

-ER NFKB1 -ER + 4-OHT -SR as

+2.5 IκBα-SR + 4-OHT WB 147 NF-κB responsive genes 8

6

4 1.2

1 2 0.8

expression (fold) 0.6 -8 -6 -4 -2 2 4 6 8

κ 0.4

NF- B target genes NFKBIA -2

0.2 4-OHT treatment expression

-4 Relative 0

-6 NF-κB target gene repression: EtOH treatment P = 1.6x10-5 4-OHT treatment -8 EtOH treatment expression

Supplementary Figure 3. Expression levels of NF-κB target genes are reduced in aged K14:NFKB1∆SP-ER skin after 4-OHT treatment. Left: The average expression level of each gene in old EtOH-treated skin (x-axis) and the contralateral 4-OHT-treated skin (y-axis) was plotted for all genes (the raw expression values are in log2 space) and shown in gray. Shown are the expression levels of several canonical NF-κB target genes (red squares), including NFKBIA, STAT5A, IRF1, PTX3, CD83, and CSF2 that were previously shown to be induced by NF-κB in primary human keratinocytes (Hinata et al., Oncogene, 2003). NF-κB target genes were coordinately repressed upon 4-OHT addition (P = 1.6x10-5, hypergeometric distribution). Dashed line indicates linear regression trend line for all genes; most red squares are below the dashed line, indicating NF-κB target gene repression after 4-OHT treatment. Right: Quantitative RT-PCR confirms repression of NFKBIA after 4-OHT treatment. PI3 kinase complex mitotic checkpoint translation regulator activity receptor protein tyrosine phosphatase signaling pathway chromatin assembly or disassembly

cell cycle

protein deubiquitination mitochondrion organization and biogenesis GO modules (n = 1308)

inflammatory response

immune response

Induced expression Repressed expression

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Expression change upon NF-κB blockade (P-value)

Supplementary Figure 4. Gene Ontology categories affected by NF-κB blockade. We used gene module map to identify coordinate induction or repression of genes that comprise each Gene Ontology (GO) category (Harris et al., Nucleic Acids Res, 2004). We calculated the average expression of all genes in each module and determined the significance of expression changes upon NF-κB blockade in old mice using Student's t-test. Red indicates that average expression of all genes in a GO category is induced upon 4-OHT addition; green indicates repression. Several GO categories that were altered upon NF-κB blockade are highlighted (P<0.1, dashed line). Genes in the immune response and inflammatory response GO terms were not significantly altered. 6

4

2

-6 -4 -2 2 4 6 Observed

-2

-4 4-OHT-induced gene expression changes: FDR>0.19 -6 Expected

Supplementary Figure 5. 4-OHT treatment has no effect on gene expression. To control for gene expression changes induced by the drug 4-OHT, we carried out the same drug/control treatment as described in the main text in duplicate on a 20 month old non-transgenic mouse that was of identical genetic background. Two-class SAM was used to identify genes that changed expression between EtOH- and 4-OHT-treated samples. Increased and decreased expression associated with 4-OHT treatment is represented as upward and downward deflections from the above plot comparing observed data in the y-axis and expected data from permutation in the x-axis. No genes were significantly induced or repressed upon 4-OHT addition (dashed lines, FDR>0.19). Cleaved Caspase-3 Newborn Young EtOH Old 4-OHT

30 µm

Supplementary Figure 6. NF-κB blockade in old skin does not induce apoptosis. Immunohistochemistry of Cleaved Caspase-3 protein expression, which detects the activated form of Caspase-3 found in cells undergoing apoptosis, in the epidermis of paraffin-embedded sections of K14:NFKB1∆SP-ER mice is shown. NF-κB blockade did not induce aberrant apoptosis in old skin. A Genes induced with senescence 0.8 (Mammary Fibroblasts) 0.6 Correlation of gene 0.4 expression to age: Skin Fibroblasts 0.2 Kidney Medulla

(expression) Kidney Cortex 2 0 20 40 60 80 100 Brain -0.2 Muscle (Abdomen)

senescence genes Muscle (Arm/Leg) -0.4 Average log -0.6 Age (years) -0.8

B

Mouse Samples: Young Old EtOH Old 4-OHT Tgfb1i4 * Rest * Pja2 * Rab14 * Akap9 * Bcl6 * Pja2 Vdp P4ha1 Nr4a3 with age in mouse Aplp2 Senescence genes induced

-1.5 +1.5

Supplementary Figure 7. Expression of several senescence markers are reversed upon NF-κB blockade in mouse skin. (A) The average expression of genes whose expression is induced after senescence initiaion in mammary fibroblasts (Hardy et al., Mol. Bio. Cell, 2005) showed consistent increase with age in six human tissues. (B) Shown are the senescence genes that showed induction with age in K14:NFKB1∆SP-ER mouse skin (P<0.05, Student’s t-test between Young and Old EtOH). Six of the eleven genes were coordinately re-silenced upon NF-κB blockade by addition of 4-OHT in old skin (*, P<0.05, Student’s t-test between Old EtOH and Old 4-OHT). al., Dev.Bio.,2006);DN-cJun inprimarykeratinocytes(Zhangetal.,CancerRes., 2007). (Silva-Vargas etal.,Dev.Cell, 2006);IRF6nullskin(Ingrahametal.,Nat.Genet., 2006);p63nullskin(Kosteret phenotypes, noneofthem affected thegenesignatureofaging.References: relative geneexpressionoftheagingsignaturegenes ±s.d.).Whileeachofthesefactorshavedemonstratable human keratinocytesinresponsetogeneticactivation ordeletionoftheindicatedtranscriptionfactor(average additional transcriptionfactors.Weanalyzedthegenomic expressionprogramsofyoungmouseskinorprimary untreated (Student's microarray experimentsisshown(±s.d.).*, average foldchangeinexpressionofthe Supplementary Figure8. A B

Aging signature (fold relative gene expression) Gene expression (fold change) 1.5 2.5 3.5 0.5 0 0 1 0 0 . . . . . 2 3 4 0 1 0 1 2 4 6 8 2 K14: K14:NFKB1 β WT

-Catenin-ER skin Young t -test). (B) Theaging geneexpressionsignatureisnotaffectedbyactivationordeletionof β -Catenin Keratin 17 Keratin 6 active Old EtOH ∆ SP (A)HyperproliferativekeratinsareinducedbyMYCbut notbyNF- -ER Old-4 OHT 0 0 0 0 1 . . . . . 0 1 2 4 6 8 2 WT 0.5 1.5 2.5 3.5 KRT2-6a IRF6 nullskin 0 1 2 3 4 P <0.05 up-regulatedgeneexpressioncomparedtowild-type

IRF6 null Untreated (Keratin6)and Wild-type

4-OHT (1d) 0 0 0 0 1 . . . . . 0 1 2 4 6 8 2 4-OHT (4d) KRT1-17 p WT 6 3

n u l 0.5 1.5 2.5 3.5 l

p63 null s 0 1 2 3 4 (Keratin17)genesfromthe k K14: i n β

Untreated K14:MYC-ER -Catenin-ER skin 0 0 0 0 1 . . . . . 0 1 2 4 6 8 2 Dominant negativecJun (primary keratinocytes) 4-OHT (1d) LacZ κ B blockade.The *

4-OHT (4d) * DN-cJun

Supplementary Table 1. Microarray data sets. Listed are the microarray experiments analyzed in this study. The age range is provided for samples used in the motif module map analysis and subsequent validation.

Description # of arrays Application Reference

Human kidney medulla (29-92 Motif module map – Figs. 1, 2, 62 [1] yrs) Table 1 Human kidney cortex (27-92 Motif module map – Figs. 1, 2, 72 [1] yrs) Table 1 Human dermal fibroblasts (17- Motif module map – Figs. 1, 2, Chang et al., 49 92 yrs) Table 1 in prep Motif module map – Figs. 1, 2, Human brain (26-106 yrs) 30 [2] Table 1 Human abdominal muscle (24- Motif module map – Figs. 1, 2, 62 [3] 83 yrs) Table 1 Human skeletal muscle, Motif module map – Figs. 1, 2, 19 [3] Arm/Leg (16-89 yrs) Table 1 Human dermal fibroblasts (8- 12 yrs normal; 8-14 yrs Motif module validation – Fig. 2, 18 [4] Hutchinson-Gilford progeria Table 1 syndrome) Motif module validation – Fig. 2, Mouse liver (6 and 22 months) 7 [5] Table 1 Mouse liver (18 month, +/- 14 Motif module validation – Fig. 2 [6] high-fat diet, +/- resveratrol) Mouse hematopoietic stem Motif module validation – Fig. 2, 8 [7] cells (2-3 and 22-24 months) Table 1 Motif module validation – Fig. 2, Mouse heart (E12.5-1 yr) 24 [8] Table 1 Overexpression of GFP vs. NF-κB p50 and p65 in primary 2 NF-κB module validation – Fig. 3 [9] human keratinocytes Overexpression of vector vs. IκBα in TNF-α treated primary 6 NF-κB module validation – Fig. 3 [10] human synovial fibroblasts Conditional NF-κB blockade in old K14:NFKB1∆SP-ER mouse skin, 14 mouse epidermis – Figs. 4, 6, This study young vs. old vs. old + 4-OHT Supp. Figs. 3, 4, 8 Layer-specific transcriptional Expression location of age- 18 [11] profiles of mouse skin associated genes – Fig. 4 Gene expression signatures of Stress signature induction by NF- 45 [12] cell stress in epithelial cells κB blockade – Fig. 4 K14:NFKB1∆SP-ER mouse skin, Conditional NF-κB blockade in 8 This study young EtOH vs. young 4-OHT young mouse epidermis – Fig. 6 Wild type mouse skin, old Induction of hyperproliferation in 8 This study acetone vs. old TPA mouse skin – Fig. 6 Wild type and K14:MYC-ER Conditional MYC induction in mouse skin, young untreated 18 mouse epidermis – Fig. 6, Supp. [13] or with 4-OHT for 1 or 4 days Fig. 8 Expression of GFP, NFKB1∆SP- Specificity of NFKB1∆SP-ER ER, or IκBα in TNF-α-induced 8 repressing TNF-α-induced NF-κB This study primary human keratinocytes target genes – Supp. Fig. 2 Wild type and K14:β-Catenin- Conditional β-Catenin activation 10 [14] ER mouse skin in mouse skin – Supp. Fig. 8

Wild type and IRF6-/- mouse 6 IRF6 null skin – Supp. Fig. 8 [15] skin Wild type and p63-/- mouse 4 p63 null skin – Supp. Fig. 8 [16] skin Expression of LacZ or DN-cJun Expression of dominant negative 2 [17] in primary human keratinocytes cJun – Supp. Fig. 8

REFERENCES

1. Rodwell, G.E. et al. A transcriptional profile of aging in the human kidney. PLoS Biol 2, e427 (2004). 2. Lu, T. et al. Gene regulation and DNA damage in the ageing human brain. Nature 429, 883-91 (2004). 3. Zahn, J.M. et al. Transcriptional profiling of aging in human muscle reveals a common aging signature. PLoS Genet 2, e115 (2006). 4. Csoka, A.B. et al. Genome-scale expression profiling of Hutchinson-Gilford progeria syndrome reveals widespread transcriptional misregulation leading to mesodermal/mesenchymal defects and accelerated atherosclerosis. Aging Cell 3, 235-43 (2004). 5. Boylston, W.H. et al. Altered cholesterologenic and lipogenic transcriptional profile in livers of aging Snell dwarf (Pit1dw/dwJ) mice. Aging Cell 3, 283-96 (2004). 6. Baur, J.A. et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444, 337-42 (2006). 7. Rossi, D.J. et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A 102, 9194-9 (2005). 8. Genomics of Cardiovascular Development, Adaptation, and Remodeling. NHLBI Program for Genomic Applications, Harvard Medical School. URL: http://www.cardiogenomics.org [May, 2007]. 9. Hinata, K., Gervin, A.M., Zhang, J.Y. & Khavari, P.A. Divergent gene regulation and growth effects by NF-kappa B in epithelial and mesenchymal cells of human skin. Oncogene 22, 1955-1964 (2003). 10. Zhang, H.G. et al. Novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis. Arthritis Rheum 50, 420-31 (2004). 11. April, C.S. & Barsh, G.S. Skin layer-specific transcriptional profiles in normal and recessive yellow (Mc1re/Mc1re) mice. Pigment Cell Res 19, 194-205 (2006). 12. Murray, J.I. et al. Diverse and specific gene expression responses to stresses in cultured human cells. Mol Biol Cell 15, 2361-74 (2004). 13. Frye, M., Gardner, C., Li, E.R., Arnold, I. & Watt, F.M. Evidence that Myc activation depletes the epidermal stem cell compartment by modulating adhesive interactions with the local microenvironment. Development 130, 2793-808 (2003). 14. Silva-Vargas, V. et al. Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells. Dev Cell 9, 121-31 (2005). 15. Ingraham, C.R. et al. Abnormal skin, limb and craniofacial morphogenesis in mice deficient for interferon regulatory factor 6 (Irf6). Nat Genet 38, 1335-40 (2006). 16. Koster, M.I., Kim, S., Huang, J., Williams, T. & Roop, D.R. TAp63alpha induces AP- 2gamma as an early event in epidermal morphogenesis. Dev Biol 289, 253-61 (2006). 17. Zhang, J.Y., Adams, A.E., Ridky, T.W., Tao, S. & Khavari, P.A. Tumor necrosis factor receptor 1/c-Jun-NH2-kinase signaling promotes human neoplasia. Cancer Res 67, 3827-34 (2007).

Supplementary Table 2. Age-associated motif modules induced in six human tissue types. Listed are the motif modules that are induced with age in human tissues (indicated by a +). Notations following some transcription factor names (ex. Q6_01) are identifiers for variants of the motifs according to TRANSFAC. Standalone sequences or sequences before a transcription factor name represent consensus binding motifs (key for combination of nucleotides: Y = C or T; R = A or G; W = A or T; S = C or G; K = T or G; M = C or A; N = unknown). Individual motifs of motif combinations are separated by an @.

Motif module: Kidney Cortex Medulla Kidney Brain (Abdomen) Muscle Muscle (Arm/Leg) Fibroblasts

AAAYRNCTG@V_NKX22_01 + [email protected] + [email protected] + ACAWNRNSRCGG + ACCTGTTG@V_FOXO1_01 + ACTAYRNNNCCCR@GGAANCGGAANY + [email protected] + + ACTWSNACTNY@V_NFY_01 + ATCMNTCCGY@CGGAARNGGCNG + CACGTG.MYC + CACGTG.MYC@V_AP2REP_01 + CAGCTG.AP.4@TGGNNNNNNKCCAR + CAGCTG.AP.4@V_T3R_Q6@V_USF2_Q6 + CAGCTG.AP.4@V_USF2_Q6 + CAGGTG.E12@V_NFE2_01 + [email protected] + CATRRAGC@V_P53_02 + CCAATNNSNNNGCG@V_HNF3_Q6_01 + CCAATNNSNNNGCG@V_TEF_Q6 + CCAWWNAAGG.SRF + [email protected] + [email protected] + CCCNNGGGAR.OLF.1@V_DR1_Q3 + + + CCCNNGGGAR.OLF.1@V_E2F1DP1_01 + CCCNNGGGAR.OLF.1@V_EGR_Q6 + CCCNNGGGAR.OLF.1@V_ETS_Q6 + CCCNNGGGAR.OLF.1@V_ETS1_B@V_TEF1_Q6 + + CCCNNGGGAR.OLF.1@V_PPAR_DR1_Q2 + + CCCNNGGGAR.OLF.1@V_STAT5A_04 + CCCNNGGGAR.OLF.1@V_TEF1_Q6 + CCCNNGGGAR.OLF.1@V_TFIII_Q6 + CCCNNGGGAR.OLF.1@V_VDR_Q6 + CCTNTMAGA@V_HNF4_Q6 + + [email protected] + CGGAARNGGCNG@GTTGNYNNRGNAAC + + [email protected] + CTGCAGY@V_CACCCBINDINGFACTOR_Q6 + CTGCAGY@V_ETS2_B + [email protected]@V_HMGIY_Q3 + CTGYNNCTYTAA@V_MRF2_01 + + CTTTGA.LEF1@V_MIF1_01 + CTTTGA.LEF1@V_MYOGNF1_01 + GATAAGR.GATA.X@V_ER_Q6_02 + + GCCATNTTG.YY1@TGCGCANK + GCCNNNWTAAR@V_HFH3_01 + [email protected] + GCGNNANTTCC.C.REL@V_NFY_01 + + GCGSCMNTTT@V_HNF1_Q6_01 + GCTNWTTGK + GCTNWTTGK@V_KROX_Q6 + [email protected] + GGAMTNNNNNTCCY@V_IRF7_01 + GGARNTKYCCA@V_AP2REP_01 + GGARNTKYCCA@V_P300_01 + + [email protected] + [email protected] + GGCNKCCATNK@V_STAT1_02 + [email protected] + GGCNNMSMYNTTG@V_NFY_01 + [email protected] + [email protected]@TTCYNRGAA.STAT5A + [email protected]@V_LHX3_01 + GGGAGGRR.MAZ@GTGGGTGK@V_ELF1_Q6 + GGGAGGRR.MAZ@V_FOXD3_01 + GGGAGGRR.MAZ@V_HMEF2_Q6 + GGGAGGRR.MAZ@V_HMGIY_Q3 + GGGAGGRR.MAZ@V_HNF3_Q6_01 + GGGAGGRR.MAZ@V_HNF3ALPHA_Q6 + GGGAGGRR.MAZ@V_ICSBP_Q6 + GGGAGGRR.MAZ@V_IRF_Q6_01 + GGGAGGRR.MAZ@V_IRF7_01 + GGGAGGRR.MAZ@V_ISRE_01 + GGGAGGRR.MAZ@V_NFAT_Q4_01 + GGGAGGRR.MAZ@V_NKX62_Q2 + GGGAGGRR.MAZ@V_PAX2_02 + GGGAGGRR.MAZ@V_PXR_Q2 + GGGAGGRR.MAZ@V_SRF_Q5_02 + GGGAGGRR.MAZ@V_STAT_Q6 + GGGAGGRR.MAZ@V_STAT4_01 + GGGAGGRR.MAZ@V_TFIIA_Q6 + [email protected] + + GGGCGGR.SP1@V_FOXO3_01 + GGGCGGR.SP1@V_FOXO4_01 + GGGCGGR.SP1@V_HMEF2_Q6 + GGGCGGR.SP1@V_OCT_Q6 + GGGCGGR.SP1@V_PAX2_02 + GGGCGGR.SP1@V_TEF_Q6 + [email protected] + GGGCGGR.SP1@YRCCAKNNGNCGC + GGGNNTTTCC.NF.KAPPAB + GGGNNTTTCC.NF.KAPPAB@TTCYRGAA + GGGNNTTTCC.NF.KAPPAB@V_MAZ_Q6 + GGGTGGRR.PAX.4@V_AFP1_Q6@V_AP2_Q6_01 + GGGTGGRR.PAX.4@V_CEBPA_01 + GGGTGGRR.PAX.4@V_ETS_Q6 + GGGTGGRR.PAX.4@V_ETS1_B + GGGTGGRR.PAX.4@V_ETS1_B@V_LFA1_Q6 + GGGTGGRR.PAX.4@V_ISRE_01 + GGGTGGRR.PAX.4@V_LFA1_Q6@V_STAT_Q6 + GGGTGGRR.PAX.4@V_LHX3_01 + GGGTGGRR.PAX.4@V_NFAT_Q4_01 + GGGTGGRR.PAX.4@V_NKX61_01 + GGGTGGRR.PAX.4@V_PU1_Q6 + GGGTGGRR.PAX.4@V_STAT6_01 + GGGYGTGNY + GGGYGTGNY@V_ETF_Q6 + + [email protected] + GTCNYYATGR@V_GATA3_01 + [email protected] + GTGGGTGK@V_EBOX_Q6 + GTGGGTGK@V_NFKB_Q6_01 + GTGGGTGK@V_RSRFC4_01 + GTTGNYNNRGNAAC@V_STAT1_02 + GTTGNYNNRGNAAC@V_TEL2_Q6 + KCCGNSWTTT@V_RFX1_02 + KCCGNSWTTT@V_SP1_Q6_01 + KCCGNSWTTT@WTTGKCTG + KRCTCNNNNMANAGC@WGTTNNNNNAAA + [email protected]@V_TFIII_Q6 + KTGGYRSGAA@V_TFIII_Q6 + [email protected] + MGGAAGTG.GABP@TMTCGCGANR + MGGAAGTG.GABP@V_ETF_Q6 + MGGAAGTG.GABP@V_STAT4_01@V_TFIII_Q6 + MYAATNNNNNNNGGC@V_RFX1_02 + RCGCANGCGY.NRF.1@V_CREBATF_Q6 + RCGCANGCGY.NRF.1@V_CREBP1CJUN_01 + RCGCANGCGY.NRF.1@V_FREAC7_01 + RGAGGAARY.PU.1@V_MYOGNF1_01 + RGAGGAARY.PU.1@V_NFKAPPAB_01 + RGAGGAARY.PU.1@V_SP1_Q6_01 + RGAGGAARY.PU.1@V_STAT1_02 + RGAGGAARY.PU.1@V_T3R_Q6 + RGTTAMWNATT.HNF.1@V_AP2_Q6_01 + RNGTGGGC@V_DR1_Q3 + RNGTGGGC@V_MZF1_01 + RNTCANNRNNYNATTW@V_AP2ALPHA_01 + + RNTCANNRNNYNATTW@V_AP2GAMMA_01 + RNTCANNRNNYNATTW@V_ETF_Q6 + RNTCANNRNNYNATTW@V_KROX_Q6 + RRAGTTGT@V_AFP1_Q6 + [email protected] + [email protected] + SCGGAAGY.ELK.1@TGCGCANK + SGCGSSAAA.E2F[email protected] + SMTTTTGT@V_HFH3_01 + SMTTTTGT@V_TCF4_Q5 + SYATTGTG@V_AP2REP_01 + TAATTA.CHX10@V_AP2_Q6_01 + TAATTA.CHX10@V_FOXO1_01 + [email protected] + TATAAA.TATA@V_IRF1_01 + TATAAA.TATA@V_SP1_Q6_01 + TCCATTKW@WTGAAAT + TGACATY@V_HNF4ALPHA_Q6 + + TGACATY@V_SMAD4_Q6 + TGACCTTG.ERRALPHA@V_AR_Q6@V_HEB_Q6 + TGACCTTG.ERRALPHA@V_LFA1_Q6 + TGANTCA.AP.1 + TGANTCA.AP.1@V_LFA1_Q6 + TGASTMAGC.NF.E2@V_DR1_Q3 + TGASTMAGC.NF.E2@V_LFA1_Q6 + TGATTTRY.GFI.1@V_PEBP_Q6 + [email protected] + TGCGCANK@V_POU1F1_Q6 + TGGAAA.NF.AT@V_NERF_Q2 + TGGNNNNNNKCCAR@V_AP2REP_01 + TGGNNNNNNKCCAR@V_LFA1_Q6@V_PU1_Q6 + TGGNNNNNNKCCAR@V_PPAR_DR1_Q2 + TGGNNNNNNKCCAR@V_SP3_Q3 + TGGNNNNNNKCCAR@V_TFIII_Q6 + TGTTTGY.HNF.3@V_AP2REP_01 + TGTTTGY.HNF.3@V_GRE_C + TMTCGCGANR@V_STAT3_02 + TMTCGCGANR@V_STAT5A_04 + TTANTCA.AP.1@V_AP2ALPHA_01 + TTANWNANTGGM@V_AHR_Q5 + TTAYRTAA.E4BP4@V_AP2_Q6_01 + TTCYRGAA@V_AP2_Q6_01 + TTCYRGAA@V_PU1_Q6 + + TTCYRGAA@V_SMAD4_Q6@V_TEF1_Q6 + TTCYRGAA@V_SP3_Q3 + TTCYRGAA@V_TEF1_Q6 + TTGCWCAAY.C.EBPBETA + TTGCWCAAY.C.EBPBETA@YAATNANRNNNCAG + [email protected] + + V_AHR_Q5 + V_AHR_Q5@V_TBP_01 + V_AHRHIF_Q6@V_TBP_01 + V_AP2_Q6_01@V_FOXO4_01 + V_AP2_Q6_01@V_HFH3_01 + V_AP2ALPHA_01@V_BRN2_01 + V_AP2ALPHA_01@V_IRF1_01@V_STAT4_01 + V_AP2ALPHA_01@V_OCT1_07 + V_AP2GAMMA_01@V_CEBPGAMMA_Q6@YKACATTT + V_AP2GAMMA_01@V_IPF1_Q4_01 + V_AP2REP_01@V_GRE_C + V_AP2REP_01@V_USF2_Q6 + V_BACH2_01@V_ETS1_B + V_BRN2_01@V_MAZR_01 + V_CACCCBINDINGFACTOR_Q6@V_LFA1_Q6@YGTCCTTGR + V_CDPCR3HD_01 + V_CEBP_Q3@V_MAZR_01 + V_CEBPA_01@V_KROX_Q6 + V_CEBPB_01@V_MAZ_Q6 + V_CEBPB_01@V_SP1_Q6_01 + V_CEBPDELTA_Q6@V_KROX_Q6 + V_CHOP_01 + V_COUP_01@V_HAND1E47_01 + V_CREBATF_Q6@V_GABP_B + V_CRX_Q4@V_SP1_Q6_01 + V_E2F_Q6_01@V_HMGIY_Q3 + V_E2F_Q6_01@WTTGKCTG + V_E2F1_Q4_01@V_NFY_01 + V_E2F1_Q4_01@WTTGKCTG + V_E2F1DP1RB_01@V_ETF_Q6 + V_E47_02@V_PPAR_DR1_Q2 + V_E47_02@V_T3R_Q6 + V_EGR_Q6@V_FREAC2_01 + V_EGR_Q6@V_FREAC3_01 + V_EGR_Q6@V_HMEF2_Q6 + V_EGR_Q6@V_ISRE_01 + V_EGR3_01@V_FOXO1_01 + V_ELK1_02 + V_ELK1_02@YTCCCRNNAGGY + V_ETF_Q6@V_FOXO1_01 + V_ETS1_B@V_SP3_Q3 + V_ETS2_B@V_HEB_Q6 + V_ETS2_B@V_MZF1_01 + V_ETS2_B@V_P53_02 + + V_FAC1_01@V_MZF1_01 + V_FAC1_01@V_NRF1_Q6 + V_FOX_Q2@V_MAZR_01 + V_FOX_Q2@YGCGYRCGC + V_FOXD3_01@V_SP1_Q6_01 + V_FOXD3_01@YGCGYRCGC + V_FOXJ2_01@V_MAZR_01 + V_FREAC7_01@V_NRF1_Q6 + V_FREAC7_01@V_USF2_Q6 + V_GABP_B + V_GRE_C@V_LFA1_Q6 + V_HFH3_01@V_KROX_Q6 + V_HFH3_01@V_MAZ_Q6 + V_HFH3_01@V_MAZR_01 + V_HFH3_01@V_SP1_Q6_01 + V_HMEF2_Q6 + V_HMEF2_Q6@V_KROX_Q6 + V_HMGIY_Q3@V_MAZR_01 + + V_HNF1_Q6_01@V_MAZR_01 + V_HNF3_Q6_01@V_MAZR_01 + V_HNF3_Q6_01@V_SP1_Q6_01 + V_HNF6_Q6@V_MAZ_Q6 + V_HNF6_Q6@V_NRF1_Q6 + V_HSF2_01@V_MZF1_01 + V_ICSBP_Q6@V_MZF1_01 + V_ICSBP_Q6@V_SP1_Q6_01 + V_IRF_Q6_01@V_MAZR_01 + V_ISRE_01@V_MAZR_01 + V_ISRE_01@V_TFIII_Q6 + V_KROX_Q6@V_MRF2_01 + V_KROX_Q6@V_PAX6_01 + V_KROX_Q6@V_PIT1_Q6 + V_KROX_Q6@YNTTTNNNANGCARM + V_LFA1_Q6@V_TEF1_Q6 + V_LXR_DR4_Q3 + V_MAZ_Q6@V_NFKAPPAB65_01 + V_MAZ_Q6@V_OCT_Q6 + V_MAZ_Q6@V_PIT1_Q6 + V_MAZ_Q6@V_SRF_Q5_02 + V_MAZ_Q6@V_STAT_Q6 + V_MAZR_01@V_PIT1_Q6 + V_MAZR_01@V_RSRFC4_01 + V_MAZR_01@V_SRF_Q5_02 + V_MAZR_01@V_TST1_01 + V_MEIS1AHOXA9_01@V_VDR_Q6 + V_MRF2_01@V_SP1_Q6_01 + V_MZF1_01@V_PXR_Q2 + V_MZF1_01@V_SRF_Q5_02 + V_NFAT_Q4_01@V_TFIII_Q6 + V_NFKAPPAB_01@V_TFIII_Q6 + V_NFKAPPAB50_01@V_STAT6_01 + V_NFKAPPAB65_01 + V_NFKAPPAB65_01@V_TFIII_Q6 + V_NFY_01@V_SREBP1_01 + [email protected] + V_NFY_01@YGCGYRCGC + V_NRF1_Q6@V_TATA_01 + V_PEBP_Q6 + V_PXR_Q2@V_SP1_Q6_01 + V_RFX1_02@V_TEL2_Q6 + V_RREB1_01@YNGTTNNNATT + V_SP1_Q6_01@V_SREBP1_01 + V_SP1_Q6_01@V_SRF_Q5_02 + V_SP1_Q6_01@V_STAT_Q6 + V_SP1_Q6_01@V_TEL2_Q6 + [email protected] + V_SREBP1_01 + V_T3R_Q6@[email protected] + + V_TCF11MAFG_01@YGCANTGCR + V_TEL2_Q6@YTCCCRNNAGGY + [email protected] + WWTAAGGC + Supplementary Table 3. Age-associated motif modules repressed in six human tissue types. Listed are the motif modules that are repressed with age in human tissues (indicated by a +). Notations following some transcription factor names (ex. Q6_01) are identifiers for variants of the motifs according to TRANSFAC. Standalone sequences or sequences before a transcription factor name represent consensus binding motifs (key for combination of nucleotides: Y = C or T; R = A or G; W = A or T; S = C or G; K = T or G; M = C or A; N = unknown). Individual motifs of motif combinations are separated by an @.

Motif module: Kidney Cortex Medulla Kidney Brain (Abdomen) Muscle Muscle (Arm/Leg) Fibroblasts

AAAYWAACM.HFH.4@V_EGR_Q6 + [email protected] + AAGWWRNYGGC@GGAANCGGAANY + [email protected] + AAGWWRNYGGC@V_NFY_01 + [email protected] + ACAWNRNSRCGG@V_NRF1_Q6 + ACTAYRNNNCCCR + [email protected] + [email protected] + ACTAYRNNNCCCR@WTTGKCTG + ACTAYRNNNCCCR@YGCGYRCGC + [email protected] + ACTWSNACTNY@V_ELK1_02 + [email protected] + + ARGGGTTAA.FXR@V_MAZ_Q6 + ARGGGTTAA.FXR@V_MAZR_01 + ARGGGTTAA.FXR@V_SP3_Q3 + [email protected] + CAGGTA.AREB6@V_ERR1_Q2 + CAGNYGKNAAA@V_NRF2_Q4 + CCANNAGRKGGC@CTGCAGY@V_TFIII_Q6 + CCCNNGGGAR.OLF.1@V_HMGIY_Q3 + CCCNNGGGAR.OLF.1@V_NKX62_Q2 + CCCNNGGGAR.OLF.1@V_TTF1_Q6 + [email protected] + CCGNMNNTNACG@V_NFY_01 + [email protected] + CCTNTMAGA@V_IRF_Q6_01 + CTTTGA.LEF1@V_MYOGNF1_01 + [email protected] + GATAAGR.GATA.X + GATGKMRGCG@V_NFY_01 + GATGKMRGCG@V_SRY_02 + GCCATNTTG.YY1@TGCGCANK + GCCATNTTG.YY1@V_SP1_Q6_01 + GCCNNNWTAAR@V_ETF_Q6 + GCCNNNWTAAR@V_FREAC7_01 + GCCNNNWTAAR@V_SP1_Q6_01 + GCGNNANTTCC.C.REL@SNACANNNYSYAGA + GCGSCMNTTT@V_SP1_Q6_01 + GGATTA.PITX2 + GGCKCATGS + [email protected] + [email protected] + GGCNNMSMYNTTG@V_NFY_01 + GGGAGGRR.MAZ@V_GATA3_01 + GGGAGGRR.MAZ@V_NKX61_01 + GGGAGGRR.MAZ@V_NKX62_Q2 + GGGAGGRR.MAZ@V_STAT6_01@YGACNNYACAR + GGGAGGRR.MAZ@V_TST1_01 + GGGCGGR.SP1@V_CEBPA_01 + GGGCGGR.SP1@V_FOX_Q2 + GGGCGGR.SP1@V_GATA6_01 + GGGCGGR.SP1@V_HNF1_Q6_01 + GGGCGGR.SP1@V_HOXA4_Q2 + GGGCGGR.SP1@V_NKX3A_01 + GGGCGGR.SP1@V_OCT_Q6 + GGGCGGR.SP1@V_POU1F1_Q6 + GGGCGGR.SP1@V_RFX1_02 + [email protected] + GGGTGGRR.PAX.4@V_FXR_IR1_Q6 + GGGTGGRR.PAX.4@V_MYCMAX_01 + GGGTGGRR.PAX.4@[email protected] + GGGYGTGNY@V_NFMUE1_Q6 + GTGACGY.E4F1@SCGGAAGY.ELK.1 + GTGACGY.E4F1@V_SOX_Q6 + GTGGGTGK@V_AREB6_02 + GTGGGTGK@V_SMAD3_Q6 + [email protected] + [email protected] + [email protected] + GTTGNYNNRGNAAC@V_GABP_B + GTTGNYNNRGNAAC@V_STAT1_02 + GTTGNYNNRGNAAC@V_TEL2_Q6 + [email protected] + [email protected] + GTTNYYNNGGTNA@YGACNNYACAR + GTTRYCATRR@TGCGCANK + GTTRYCATRR@V_ELK1_02 + KCCGNSWTTT@V_SP1_Q6_01 + [email protected]@V_TFIII_Q6 + MGGAAGTG.GABP@RYTGCNWTGGNR + MGGAAGTG.GABP@TCCCRNNRTGC + RCGCANGCGY.NRF.1@V_ARNT_01 + RNGTGGGC@V_AP3_Q6 + RRAGTTGT@V_AFP1_Q6 + RTAAACA.FREAC.2@V_AP2REP_01 + RYTGCNWTGGNR@V_CDX_Q5 + [email protected] + SCGGAAGY.ELK.1@V_E2F1DP1_01 + SGCGSSAAA.E2F.1.DP.2@V_ALPHACP1_01 + SGCGSSAAA.E2F.1.DP.2@V_ICSBP_Q6 + TAANNYSGCG@V_ETF_Q6 + TAANNYSGCG@V_SP1_Q6_01 + TATAAA.TATA@V_AP2ALPHA_01 + TCCATTKW@WTGAAAT + TCCCRNNRTGC@V_GABP_B + TGACAGNY.MEIS1@V_RFX1_02 + TGACATY@V_EBOX_Q6 + TGACATY@V_SMAD4_Q6 + TGACCTY.ESRRA@V_AP2REP_01 + TGACGTCA.ATF3@V_HNF6_Q6 + TGACGTCA.ATF3@V_RFX1_02 + TGANNYRGCA.TCF11.MAFG@V_AR_Q6 + TGCCAAR.NF.1@V_EFC_Q6 + TGCTGAY@WWTAAGGC + TGGNNNNNNKCCAR@V_LFA1_Q6@V_PU1_Q6 + TGGNNNNNNKCCAR@V_TTF1_Q6 + TGTYNNNNNRGCARM@V_MEF2_04 + TGTYNNNNNRGCARM@V_MYB_Q5_01 + TMTCGCGANR@V_KROX_Q6 + TTANWNANTGGM + TTANWNANTGGM@V_AP2ALPHA_01 + TTANWNANTGGM@V_RFX1_02 + TTCYRGAA@V_ELF1_Q6 + TTGCWCAAY.C.EBPBETA + [email protected] + V_AFP1_Q6@V_AP2ALPHA_01 + V_AFP1_Q6@V_ETF_Q6 + V_AHRHIF_Q6@V_FOX_Q2 + V_AIRE_01@V_EGR_Q6 + V_AIRE_01@V_ETF_Q6 + V_AIRE_01@V_KROX_Q6 + V_ALPHACP1_01@V_E2F_Q6_01 + V_AMEF2_Q6@V_AP2ALPHA_01 + V_AMEF2_Q6@V_EGR_Q6 + V_AP2_Q6_01@V_CRX_Q4 + V_AP2_Q6_01@V_FOXO4_01 + V_AP2_Q6_01@V_NKX61_01 + V_AP2_Q6_01@V_NKX62_Q2 + V_AP2_Q6_01@V_POU1F1_Q6@V_STAT5A_04 + V_AP2_Q6_01@V_POU3F2_01 + [email protected] + V_AP2ALPHA_01@V_TATA_01 + V_AP2GAMMA_01@V_CDC5_01 + V_AP2REP_01@V_E47_02 + V_ATF_01@V_NFY_01 + [email protected] + V_CDX_Q5 + V_CDX_Q5@V_MAZ_Q6 + V_CDX_Q5@V_MAZR_01 + V_CREB_Q3 ++ V_CREBATF_Q6@V_SOX_Q6 + + V_CREBP1CJUN_01@V_HNF6_Q6 + V_E2F_Q6_01@V_NFY_01 + V_E2F1_Q4_01@WTTGKCTG + + V_E2F1DP1_01@V_HMGIY_Q3 + V_E2F1DP1RB_01@WTTGKCTG + V_E2F1DP2_01@WTTGKCTG + V_E47_02@V_SMAD3_Q6 + V_EGR_Q6@V_FOX_Q2 + V_EGR_Q6@V_HMEF2_Q6 + V_EGR3_01@V_HNF3_Q6_01 + V_ETF_Q6@V_FREAC3_01 + V_ETF_Q6@V_FREAC7_01 + V_ETF_Q6@V_NKX61_01 + V_ETF_Q6@V_NKX62_Q2 + V_ETF_Q6@V_PIT1_Q6 + V_ETF_Q6@V_POU3F2_01 + V_ETS_Q6@YTCCCRNNAGGY + V_FOXJ2_01@V_MTF1_Q4 + V_FREAC3_01@V_KROX_Q6 + V_GATA6_01 ++ V_HES1_Q2 + V_HIF1_Q3 + V_HMEF2_Q6@V_SP1_Q6_01 + V_HNF1_Q6_01@V_MAZ_Q6 + V_KROX_Q6@V_NKX3A_01 + V_KROX_Q6@V_PAX2_02 + V_KROX_Q6@V_POU3F2_01 + V_LFA1_Q6@[email protected] + V_LHX3_01@V_NFY_01 + V_LMO2COM_02 + V_MAX_01@V_RORA2_01 + V_MAZ_Q6@V_NKX3A_01 + V_MAZ_Q6@V_TBP_01 + [email protected] + V_MAZR_01@V_POU1F1_Q6 + V_MAZR_01@V_RSRFC4_01 + V_MAZR_01@V_TATA_01 + [email protected] + V_MEIS1_01@V_PPARG_01 + V_MTF1_Q4 + V_NFY_01@V_SREBP1_01 + V_NRSF_01 + V_OCT1_07 + V_PBX1_02 + V_POU1F1_Q6@V_SP1_Q6_01 + + V_POU3F2_01@V_SP1_Q6_01 + V_RFX_Q6 + V_RORA1_01 + V_RREB1_01@V_SMAD_Q6_01 + V_SP1_Q6_01@V_TATA_01 + [email protected] + V_SREBP1_01 + V_STAT4_01@YTCCCRNNAGGY + + V_TAL1ALPHAE47_01@YTAAYNGCT + V_TCF11_01 + V_TEL2_Q6@YTCCCRNNAGGY + V_TFIIA_Q6 + [email protected] + Supplementary Table 4. Age-associated and “rejuvenated” genes. Genes whose expression significantly differs between young and EtOH-treated old samples are listed in the order presented in Figure 4C, which was obtained through unsupervised hierarchical clustering. “Rejuvenated” genes show a significant difference in expression between 4-OHT- and EtOH-treated old samples. Motif module mapping of the mouse genome was used to identify the subset of genes with NF-κB binding sites (+). This gene list was intersected with layer-specific transcriptional profiles of the skin (April and Barsh, Pigment Cell Res., 2006), and the location each gene is normally expressed is provided (see figure below for key). EST, expressed sequence tag.

Clone ID Gene Symbol UniGene ID LocusLink ID “Rejuv?” κB site Location 1190031J09 Hsd17b7 Mm.12882 15490 H3115E07 Rkhd3 Mm.306789 108797 SE+BE 1810022N05 Lsp1 Mm.234003 16985 + 1190020E13 Aspn Mm.383216 66695 + IW:185 EST 1100001C17 Col1a2 Mm.277792 12843 + DM 1200014H15 Col1a2 Mm.277792 12843 + DM UI-M-AO0-acf-d-10-0-UI Col1a1 Mm.277735 12842 DM 2310045A05 Col1a1 Mm.277735 12842 DM IMAGE:575740 Ear2 Mm.327088 13587 2010307E17 EST 1190024B01 Sfrp2 Mm.19155 20319 + DM 1500019H08 Tinf2 Mm.280717 28113 + 2410002F21 EST Mm.379107 SE+BE 2810464O09 EST 15902 + BE+DM H3120E06 Smoc1 Mm.273295 64075 + H3067F07 Qk Mm.262294 19317 + H3056D10 5330438I03Rik Mm.101743 381325 Yes + 1700022L10 Dusp13 Mm.317764 27389 Yes + 1500009L16 1500009L16Rik Mm.271188 69784 Yes 1500031E24 Aldh1a1 Mm.250866 11668 + H3004F07 4931423N10Rik Mm.314026 70981 BE H3126D09 4931423N10Rik Mm.314026 70981 BE 1110019M03 Ptp4a2 Mm.193688 19244 BE H3126G10 E2f6 Mm.23296 50496 + BE 2010002D23 Ptx3 Mm.276776 19288 Yes + 1500017O22 EST Yes 1500032O06 Ccng1 Mm.2103 12450 Yes + BE H3077H03 Ccng1 Mm.2103 12450 Yes + BE IW:676 EST H3038D03 D4Ertd89e Mm.27184 52076 Yes H3025B12 D3Jfr1 Mm.277713 229663 Yes + L0300F05 EST Yes H3071C07 D030074E01Rik Mm.386824 319936 Yes J0029A05 EST Yes SE+BE H3009D07 4930572I07Rik Mm.331576 319885 Yes SE+DM H3028A01 Atrx Mm.10141 22589 Yes H3142H02 2610312F20Rik Mm.322881 70461 Yes H3100F02 EST Mm.351134 Yes 2010307K20 1500011J06Rik Mm.276341 208606 Yes 2410008D05 Atic Mm.38010 108147 Yes + BE H3119F07 EST Yes G0120D04 2610033H07Rik Mm.40292 75416 Yes + SE+BE 2210004F16 Vars2l Mm.32002 68915 Yes 3200002N09 5730406I15Rik Mm.30043 66624 Yes SE+BE 2810004M04 Hspcb Mm.2180 15516 Yes + BE H3042G07 Hspcb Mm.2180 15516 Yes + BE G0116C08 Adora1 Mm.298908 11539 Yes + H3049H04 Hspcb Mm.2180 15516 Yes + BE IW:1320 EST Yes H3073G06 Tsc1 Mm.224354 64930 Yes + 2510009L21 EST Yes DM H3058F09 Lphn2 Mm.9776 99633 Yes SE H3083G06 EST Mm.372431 Yes BE UI-M-AI1-afs-g-10-0-UI Notch4 Mm.173813 18132 Yes + SE+DM 2210008C21 Vapb Mm.260456 56491 + H3128G06 AW050020 Mm.262056 268420 Yes + H3048C02 EST Yes H3051G02 Cpeb3 Mm.360051 208922 Yes + UI-M-BH0-ajw-b-05-0-UI Cpeb3 Mm.360051 208922 Yes + IMAGE:819075 Flt1 Mm.3464 14254 Yes BE H3002G06 Prss32 Mm.386797 69814 Yes 2810003C01 EST Yes 2310047N07 Msl2 Mm.335386 77853 Yes SE+BE H3145B02 A930017E24Rik Mm.258142 319945 + SE+BE IMAGE:477537 Hoxc6 Mm.4444 15425 Yes + SE+BE 1500001I14 Ppp1r3c Mm.24724 53412 Yes + H3004E02 Cdkn1b Mm.2958 12576 Yes H3056G12 Pdlim5 Mm.117709 56376 Yes + SE+BE H3034F12 EST Yes H3036D06 EST Yes H3036D08 Mdn1 Mm.24627 100019 Yes J0028D11 Catns Mm.35738 12388 Yes + IW:212 EST Yes IMAGE:324054 EST 4803 Yes SE+BE IW:518 EST Yes H3053H09 EST Yes H3052D04 EST Yes H3056F11 A830023L05 Mm.236368 270049 Yes H3056B01 EST Yes H3056F09 EST Yes IW:507 EST Yes IW:77 EST Yes H3055D06 EST Yes H3052B12 EST Yes H3139C10 EST Yes IW:470 EST Yes IW:570 EST Yes IW:550 EST Yes H3119B01 EST Yes H3132A10 EST Yes H3116F10 Rbm16 Mm.124373 106583 Yes IW:191 EST Yes H3112E03 EST Yes H3074B02 EST Yes 2410003E21 Amhr2 Mm.60331 110542 Yes H3154E01 Rad50 Mm.4888 19360 Yes + H3028A11 Smc2l1 Mm.2999 14211 Yes + SE+BE IW:317 EST Yes 2610101K02 EST Mm.326577 Yes 1810008M12 EST Mm.280968 Yes BE H3009F01 Rrbp1 Mm.13705 81910 Yes DM 2610019M15 Ptpn11 Mm.8681 19247 Yes + 2310043D08 2310043D08Rik Mm.202518 76464 Yes + H3036G03 BC026657 Mm.237935 208618 Yes H3070G07 Gpr48 Mm.277351 107515 Yes IW:1469 EST Yes H3038D06 9130404D08Rik Mm.269061 74549 Yes + H3134H05 Jarid1a Mm.288602 214899 Yes 2810417K24 Smc2l1 Mm.2999 14211 Yes + SE+BE 2610029M07 Tpr Mm.174256 108989 Yes BE H3059A04 4632413K17Rik Mm.295246 216440 Yes + SE+BE H3065G11 Selp Mm.3337 20344 Yes + IW:1028 EST Yes 2610527L21 EST Yes 1810044B15 4632413K17Rik Mm.295246 216440 Yes + SE+BE 2300002F20 Smc6l1 Mm.173953 67241 Yes + SE+BE H3059B05 Acbd3 Mm.272981 170760 Yes SE H3040G06 Eif4g1 Mm.260256 208643 Yes + 3110002L24 Nvl Mm.263464 67459 Yes SE+BE 2310011F09 Alas2 Mm.302724 11656 Yes + SE H3067H08 2610101N10Rik Mm.292742 67958 Yes + BE H3049E03 Dnajc2 Mm.266312 22791 Yes IW:1368 EST Yes 2310033G13 EST 54214 Yes + SE 1700007M11 5730406M06Rik Mm.100117 66625 Yes 1.50003E+11 EST 17311 Yes + H3071A12 Stk25 Mm.28761 59041 Yes + 2810404I24 1700009P03Rik Mm.291542 74213 Yes + SE+BE H3040A12 Ctcf Mm.269474 13018 Yes + BE H3004A06 Ttc3 Mm.213408 22129 Yes H3037A05 Golga4 Mm.10409 54214 Yes + SE 1810015O06 Srpr Mm.284688 67398 Yes + 1110037C24 EST Yes IW:967 EST Yes H3066E04 Ppig Mm.11815 228005 Yes H3087H03 5730555F13Rik Mm.22379 66660 Yes + H3127F03 5730407K14Rik Mm.295647 101113 Yes + SE+BE 2210008A20 EST 235533 Yes + H3084G12 Suv420h1 Mm.278578 225888 Yes H3069C09 Rock1 Mm.6710 19877 Yes + BE+DM H3142B04 BC037112 Mm.286885 231128 Yes BE IW:1384 EST Yes UI-M-AM0-adu-b-07-0-UI Pja2 Mm.41711 224938 Yes SE 2900006N19 Man1b Mm.271617 17156 Yes SE+BE 2610024D04 2310030G06Rik Mm.273375 66952 Yes H3109G09 Pcp4l1 Mm.273087 66425 Yes BE 2310008J09 EST Yes H3089B10 Zfp398 Mm.169059 272347 Yes H3091G03 Ubr1 Mm.330516 22222 Yes H3141C03 Tpr Mm.174256 108989 Yes BE H3045G06 Tpr Mm.174256 108989 Yes BE H3027C09 3010027A04Rik Mm.31567 77087 Yes H3023B10 Atrx Mm.10141 22589 Yes H3027D08 Nupl1 Mm.258051 71844 Yes + BE J0020D04 Adam17 Mm.27681 11491 Yes + 1110004H10 EST 71781 Yes 3110006H12 EST Yes 2310010I16 2310010I16Rik Mm.18110 66371 Yes + SE 2310040O13 5730455P16Rik Mm.86860 70591 Yes H3042H07 Smarcc1 Mm.85410 Yes BE H3042D09 Akap9 Mm.46044 100986 Yes J0028F08 Sdccag33 Mm.102136 110796 Yes UI-M-AM1-afu-c-07-0-UI Wdr42a Mm.159915 98193 Yes 2310012H03 EST Yes SE 2310012M01 EST Yes SE+BE 2310038L10 EST Yes SE 2210401K01 2210401K01Rik Mm.298256 72289 Yes SE H3109A04 2210401K01Rik Mm.298256 72289 Yes SE H3010H10 2210401K01Rik Mm.298256 72289 Yes SE IW:105 EST Yes J0417F04 Smc6l1 Mm.173953 67241 Yes + SE+BE H3063G03 EST Yes H3009D12 Nid1 Mm.4691 18073 Yes + DM UI-M-AM1-afu-e-07-0-UI Rora Mm.267116 19883 Yes H3051G12 EST Yes J0089G07 Epb4.1l4b Mm.28217 54357 Yes BE H3040F06 Mpv17 Mm.999 17527 Yes H3131H05 Ppm1l Mm.40577 242083 Yes + SE 2610020J22 EST Yes 1700008H16 EST 69288 Yes 4631405H01 EST 214547 Yes 1020009K15 Slc2a4 Mm.10661 20528 Yes + 2310038O09 Ttn Mm.373672 22138 Yes BE+DM 1500038G10 BC054438 Mm.34379 407831 Yes + H3112D02 1200007D18Rik Mm.30496 67458 + 1190031P23 Pde2a Mm.247564 207728 Yes + H3153E01 Syne2 Mm.26652 319565 Yes 2610001M21 EST Mm.385221 Yes 2310026F11 EST 67484 Yes + 2310031A19 2310005P05Rik Mm.112977 67484 Yes + H3048B01 Fubp1 Mm.278922 51886 Yes + 3110065A12 Atrx Mm.10141 22589 Yes 2210003G19 EST Yes 0610042P09 Gstk1 Mm.267014 76263 Yes + BE 2310021I05 BC064011 Mm.336856 407790 Yes 0610010I24 Eno3 Mm.251322 13808 Yes + DM 2610024M03 EST Mm.220761 Yes IW:1200 EST Yes 2610314B04 Angpt1 Mm.309336 11600 Yes + 1700020G04 EST 75552 Yes + SE 1200016O17 EST Mm.384628 Yes SE+BE H3020A08 Hip2 Mm.319512 53323 Yes SE+BE 2510010F10 Cdv3 Mm.261025 321022 + DM H3088E04 Csnk1a1 Mm.26908 93687 Yes SE+BE H3091D05 EST Mm.281895 H3060A05 Crim1 Mm.311912 50766 H3115C05 2410003B16Rik Mm.29933 72333 BE+DM H3133F03 Mtus1 Mm.149438 102103 + SE+BE 3010002B20 Pctk1 Mm.102574 18555 H3073C09 EST Yes H3021B04 Sypl Mm.246304 19027 Yes SE+BE H3155D09 Abl2 Mm.329515 11352 H3071A05 EST Yes H3099A11 EST Mm.355161 Yes H3128C04 Smad4 Mm.100399 17128 + C0186G05 Samd8 Mm.102765 67630 + 1810031L19 EST IW:1356 EST Yes J0089F06 EST Yes H3048C04 Morf4l1 Mm.379029 21761 Yes BE+DM 5730507I23 EST H3151D03 Kif1b Mm.252497 16561 Yes H3159B02 Glce Mm.24411 93683 UI-M-BH0-ajv-d-09-0-UI 2810013M15Rik Mm.45748 67493 SE+BE 2610028J06 EST 22380 2010315F03 1500011J06Rik Mm.276341 208606 H3057D04 Tbc1d15 Mm.22252 66687 Yes + 2810408M09 EST 76367 Yes H3009D06 EST Mm.259036 403194 2810453K23 Prpf4b Mm.10027 19134 + BE+DM 1810005G02 EST SE+BE H3070A07 Ppil4 Mm.38927 67418 Yes + SE+BE 2610018B07 EST 67418 Yes + SE+BE H3002F06 Eif3s10 Mm.2238 13669 C0109H08 Smc1l1 Mm.26412 24061 Yes 1500011I14 Zadh1 Mm.246127 77219 + H3055E05 Tax1bp1 Mm.50424 52440 SE H3064F12 Cbl Mm.266871 12402 + SE+BE 2310028C20 EST 24061 IW:1472 EST IW:216 EST Yes IW:204 EST Yes IW:320 EST H3018D11 Fnbp3 Mm.257474 56194 Yes H3037C09 Usp7 Mm.295330 252870 Yes 2610029I21 Sf3b2 Mm.196532 319322 Yes + BE 1810028A11 Xrn2 Mm.3065 24128 Yes + BE IMAGE:534270 Rad50 Mm.4888 19360 Yes + 2810410K23 Eif3s10 Mm.2238 13669 Yes 2610033H07 2610033H07Rik Mm.40292 75416 Yes + SE+BE H3080G10 Apc Mm.7883 11789 Yes + H3079C02 Atrx Mm.10141 22589 Yes 2810410C14 2810410C14Rik Mm.44608 230582 Yes + H3146E12 Sfrs2ip Mm.324474 72193 Yes BE H3132G02 App Mm.277585 11820 Yes SE+BE 4921503G23 EST Yes H3004D10 Eif3s10 Mm.2238 13669 Yes 2810409K13 EST 12988 Yes 2810408K05 Wtap Mm.275521 60532 Yes BE+DM H3070B08 EST Yes H3070B07 Smc1l1 Mm.26412 24061 Yes 2610027I02 Nol8 Mm.258915 70930 Yes H3007H12 Ktn1 Mm.3110 16709 Yes SE+BE H3148H10 Bclaf1 Mm.294783 72567 + 2310009O19 Kinectin1(Ktn1),mRNMm.3110 16709 Yes SE+BE 2310009K19 Mtdh Mm.130883 67154 Yes + DM J0029A07 D15Ertd366e Mm.33207 65970 Yes 2510008H13 Itih2 Mm.182043 16425 Yes SE+DM 2610031L17 EST 68879 Yes H3072G10 Ppig Mm.11815 228005 Yes H3024D06 4921537D05Rik Mm.296971 77048 + 2610002D01 Ilf3 Mm.20935 16201 Yes SE+BE 2510017G15 EST SE+BE 2510040F10 Cenpc1 Mm.4649 12617 + SE IW:1386 EST UI-M-AM1-afu-d-12-0-UI Ndufb2 Mm.29415 68198 + IW:769 EST UI-M-AJ0-aaz-d-12-0-UI Usp16 Mm.152941 74112 BE H3015E04 Trip12 Mm.209265 14897 IW:463 EST H3006D11 Suz12 Mm.283410 52615 IW:544 EST H3095B03 EST 2410004K19 2810013M15Rik Mm.45748 67493 SE+BE 2300010L08 EST 18949 SE+DM 2310031K04 Tax1bp1 Mm.50424 52440 SE H3036F09 Top2a Mm.4237 21973 + BE+DM 2510001I21 Pa2g4 Mm.4742 18813 + SE+BE H3037F03 Cab39 Mm.26135 12283 2810401D19 Nipsnap1 Mm.293716 18082 + BE IW:76 EST H3155H10 Sycp3 Mm.297977 20962 IW:1282 EST IW:107 EST H3032H05 EST 2010016J09 Srrm1 Mm.1963 51796 IW:328 EST IW:1480 EST H3104G11 G6pc3 Mm.22385 68401 + IW:1342 EST IW:305 EST IW:447 EST IMAGE:583233 Pdha1 Mm.34775 18597 BE+DM L0204B09 EST H3032G12 2900097C17Rik Mm.324242 347740 1810005K14 2900097C17Rik Mm.324242 347740 IW:1355 EST IW:1045 EST IW:417 EST IW:502 EST IW:203 EST H3032H06 EST 5730465K11 Ttf1 Mm.252195 22130 + H3041A09 Kpna3 Mm.25548 16648 + 2210416H05 EST 18673 + 1500031L05 EST H3090G02 EST Mm.382079 1500010A16 6620401M08Rik Mm.386823 75744 H3027E03 Apob Mm.221239 238055 H3027F01 EST Yes IW:933 EST Yes 2310047H24 Rbp7 Mm.46023 63954 Yes 2510018P04 EST 2510012J08 2510012J08Rik Mm.87628 70312 SE 1600024E15 EST 1020009C21 Ywhae Mm.234700 22627 Yes 1020009M15 Sec14l1 Mm.272312 74136 + SE+DM 2510028J08 EST 15129 + BE+DM H3152D06 Alas2 Mm.302724 11656 Yes + SE 1020011I17 EST Mm.139418 1600013K09 Hbb-b1 15129 + BE+DM 2510040L04 Golph3 Mm.250936 66629 + BE+DM 3200002K15 Col3a1 Mm.249555 12825 + BE+DM H3122H12 Hba-a1 Mm.196110 15122 + BE+DM 1600012O19 Hbb Mm.387214 15127 BE+DM H3118D12 Ches1 Mm.341972 71375 + 2810404B17 EST Mm.371779 BE+DM 1020013I05 Hbb-b1 Mm.288567 15129 + BE+DM UI-M-AO0-acf-f-05-0-UI Hbb-b1 Mm.288567 15129 + BE+DM H3080H05 EST 2410006A12 Hba-a1 Mm.196110 15122 + BE+DM 2510015C01 Nt5c3 Mm.158936 107569 SE 2500002D04 EST 2510049I19 2510049I19Rik Mm.28327 67922 + BE+DM 1020007M17 Hbb-b1 Mm.288567 15129 + 2700038O11 Hbb-a1 15122 + BE+DM 2210420C02 Hbb-b1 Mm.288567 15129 + BE+DM 2510018D09 EST 1110017P09 Gpr155 Mm.241098 68526 UI-M-AM1-afv-h-05-0-UI Hbb-b1 Mm.288567 15129 + 1600000O13 EST BE+DM 1600019A13 Hbb-b1 Mm.288567 15129 + BE+DM 2510023M22 EST 15129 + BE+DM 2510018N13 Hbb-b1 Mm.288567 15129 + BE+DM H3032C06 Fsd1 Mm.270660 240121 + G0120G03 EST 1600016N05 Hbb-b1 15129 + BE+DM J0073B10 EST L0072C01 EST H3126H01 Hba-a1 Mm.196110 15122 + BE+DM 3300001P16 Hbb-b1 Mm.288567 15129 + BE+DM 2510040K10 Hbb-b1 Mm.288567 15129 + BE+DM 2510044F14 Hbb-b1 15129 + BE+DM 2510040I07 Hbb-b1 15129 + BE+DM H3045B11 1700006J14Rik Mm.117464 321010 + SE+BE H3126A10 Hba-a1 Mm.196110 15122 + BE+DM 2510027H11 EST 73170 H3118D11 EST L0035C02 Hbb-b1 Mm.387214 15127 BE+DM 1600019I13 Hbb-b1 Mm.288567 15129 + BE+DM H3122H09 Hba-a1 Mm.196110 15122 + BE+DM L0223A07 Msh4 Mm.272226 55993 + L0009G02 EST H3123A02 Hba-a1 Mm.196110 15122 + BE+DM H3122H10 Hba-a1 Mm.196110 15122 + BE+DM H3125H11 Hba-a1 Mm.196110 15122 + BE+DM H3122H11 Hba-a1 Mm.196110 15122 + BE+DM H3140G04 Hba-a1 Mm.196110 15122 + BE+DM L0036G12 EST 2510022E24 2700085E05Rik Mm.249700 67201 2810001B11 EST H3123E05 EST H3123G10 Mgat1 Mm.196933 17308 BE+DM H3045A12 Hba-a1 Mm.196110 15122 + BE+DM 2600002O09 Hba-a1 Mm.196110 15122 + BE+DM H3118C11 Hba-a1 Mm.196110 15122 + BE+DM H3120F02 Pcyox1 Mm.30849 2400002B20 EST BE+DM 2510022J06 Hbb-b1 15129 + BE+DM 2810404A19 Hbb-b1 Mm.288567 15129 + BE+DM 2510012E24 Ermap Mm.290753 27028 H3117B08 EST H3123A01 Hba-a1 Mm.196110 15122 + BE+DM H3112E02 Hba-a1 Mm.196110 15122 + BE+DM L0077A03 EST H3117D02 Hbb-b1 Mm.288567 BE+DM 2510047K14 Hba-a1 Mm.196110 15122 + BE+DM 2510030M07 EST 12583 + BE L0060H12 EST 2610002L16 Hbb-bh1 Mm.378935 15132 + BE+DM 1110019H17 Kif21b Mm.276135 16565 + DM 1110053L05 EST 2310008H19 EST 66102 + BE+DM UI-M-AO0-aca-g-05-0-UI 4931423N10Rik Mm.314026 70981 BE 2.51002E+17 EST 1600022P06 BC064011 Mm.336856 407790 SE 1190014N05 BC064011 Mm.336856 407790 SE 1110054A17 Sncg Mm.282800 20618 + SE+DM IMAGE:479269 Itpr3 Mm.328900 16440 BE IW:943 EST IW:787 EST 1600002000 EST IW:849 EST Supplementary Table 5. Candidate NF-κB dependent genes that enforce aging. Of the 32 genes identified through a genome-wide RNAi screen to extend C. elegans lifespan (Curran et al., PLoS Genetics, 2007) with human and mouse orthologs/homologs, seven have evidence suggesting their regulation by the NF-κB pathway in mammals.

C. elegans longevity gene (human/mouse homologue) Evidence atp-2 (Atp5b) Present in NF-κB motif module egl-45 (Eif3s10) "Rejuvenated" by NF-κB blockade Three eukaryotic translation initiation factors "rejuvenated" (Eif3s10, eif-3.F (Eif3s5) Eif2s3y, Eif4g1) Three eukaryotic translation initiation factors "rejuvenated" (Eif3s10, eif-3.B (Eif3s9) Eif2s3y, Eif4g1) Three eukaryotic translation initiation factors "rejuvenated" (Eif3s10, inf-1 (Eif4a2) Eif2s3y, Eif4g1) W07E6.1 (Nol1) Nucleolar protein Nol14 "rejuvenated" D1054.14 (Prpf38a) pre-mRNA factor Prpf6 "rejuvenated"