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Papadaki Et Al., 2009 Supplementary

Papadaki et al., 2009 Supplementary

Supplemental Data Index

x Supplemental Figures 1-6

x Supplemental Tables 1a, 1b, 2 Papadaki et al., 2009 Supplementary

Supplemental Figure 1. Thymocyte restricted inactivation of the Elavl1 . + fl (A) Diagrammatic representation of the wild-type (Elavl1P P), floxed (Elavl1P P) and Cre- - recombined (Elavl1P P) Elavl1/HuR loci on mouse 8; Noted are the loxP sequences (triangles) flanking the selection marker (neo) used in targeting and the ATG containing exon 2 (white box); (H) denotes restriction sites for loci mapping. (B) Detection of native (+), targeted (fl) and Cre-recombinant (-) loci in thymocyte DNA extracts from control and test mice following HindIII digestion and Southern blotting. (C) Western blot of total thymic extracts probed with ĮHuR Ab + fl/fl indicating the loss of HuR protein in LckCreP PElavl1P P thymi. Į- is shown for quantitation. (D) Flow cytometric detection of intracellular mHuR protein in + fl/+ LckCreP PElavl1P P thymocytes (open histogram), and its respective loss in + fl/fl LckCreP PElavl1P P thymocytes (shaded histogram). The dotted histogram depicts the + isotype-matched background staining. (E) Flow cytometric detection of HuRP P or - + + + fl/+ HuRP P cells in gated splenic CD4P Por CD8P P T-cells from 8 week old LckCreP PElavl1P + fl/fl - Pand LckCreP PElavl1P P mice respectively. (F) Enumeration of HuRP P cells in + fl/fl LckCreP PElavl1P P thymocyte subsets and splenic T-cells; Data are percentages (+SEM) derived from the flow cytometric detection of HuR- cells in CD4/CD8/DP and DN gated populations (n=12-15) at 8-10 weeks of age. Papadaki et al., 2009 Supplementary

- Supplemental Figure 2. The increased expansion of HuRP PDN relates to - increases in immature thymocytes and the exclusive presence of HuRP PT- lymphocytes in mutant mice. (A) Detection of DN-descendant immature hi lo + lo + fl/+ CD24P PCD5P PSP cells in CD8P PTCRȕP Pgated LckCreP PElavl1P P(open + fl/fl histogram) and LckCreP PElavl1P Pthymi (filled histogram) and their respective + lo counts (+SD) as CD8P PTCRȕP P immature cells; (*)p,0.05. (B)Contour plots - + showing the representation of HuRP P and HuRP P cells expressing surface TCRȕ + fl/+ + fl/fl in gated LckCreP PElavl1P Pand LckCrePPElavl1P PDP and SP thymocytes as well as splenic T cells. Papadaki et al., 2009 Supplementary

Supplemental Figure 3. Effects of HuR deletion on the presence of TCR- + + + + restricted thymocytes (A) Detection of HuR in F5P P LckCreP PElavl1fl/P PandPPF5P P + fl/fl LckCreP PElavl1P Pthymocytes expressing the F5-TCR (Vȕ11). (B) Detection of F5 + + transgenic cells as Vȕ11-gated CD4/8 thymocytes or splenic CD8P P T cells in F5P P + fl/fl LckCreP PElavl1P Ptransgenic mice, relative to the corresponding control subsets. + + + fl/fl (C) F5P P T-cell counts in thymi and spleens of F5P P LckCreP PElavl1P Pmice relative controls. Data (+SD) derived from n=7 mice/group at 8 weeks of age; (*)underlined numbers are p values of statistical difference. (D) Detection of HuR in female + + fl/fl tgHYP P LckCreP PElavl1P Ptransgenic thymocytes or splenocytes, expressing the HY- + + fl/+ TCR (T3.70) relative to the corresponding tgHYP P LckCreP PElavl1P Pcontrols. (E) + + Detection of HY-TCRP P transgenic cells as T3.70-gated DN, DP and CD8P PSP + + + fl/fl thymocytes as well as CD8P P splenocytes in female tgHYP P LckCreP PElavl1P Pmice + + relative to the corresponding controls.(F) HY-TCRP P T-cell counts in female tgHYP P + fl/fl LckCreP PElavl1P Pthymi and spleens relative to control values. Data (+SD) from n=7 mice/group at 10 weeks of age; (*) underlined numbers correspond to p values. Papadaki et al., 2009 Supplementary

Supplemental Figure 4. Changes in the presence of chemokine - responsive HuRP Pthymocyte subsets are not due to defects in the expression of chemokine receptors. (A) qRT-PCR detection of S1PR1R (EDG- 1), Klf2, CXCR4 and CCR7 mRNAs in thymocyte extracts derived from + fl/fl LckCreP PElavl1P Pmice. Black bar graphs denote expression values (+SD) in + fl/fl LckCreP PElavl1P Pcultures as fold change relative to those from + fl/+ LckCreP PElavl1P Pcontrols (White Bar-Ref). (B) Intracellular flow cytometry histograms showing the expression of S1PR1R (EDG-1), Klf2, CXCR4 or CCR7 + + + fl/+ in gated CD4P P and CD8P P SP thymocytes of LckCreP PElavl1P P(open + fl/fl histogram) and LckCreP PElavl1P P(filled histogram) thymocytes. Dashed histograms refer to isotype match stainings. (C) Representative histograms + fl/+ showing that intracellular HuR levels in SP subsets from LckCreP PElavl1P

Pthymi are not altered between resting (open histograms) and chemokine treated (filled histograms) cells. Papadaki et al., 2009 Supplementary

8

- Supplemental Figure 5. Differences in HuRP P thymocyte deletion following antigenic or corticosteroid stimulation and detection of death-related + fl/+ mRNAs. (A) Flow cytometric detection of thymocyte loss in LckCreP PElavl1P + fl/fl Pand LckCreP PElavl1P Pmice following the in vivo injection of ĮCD3 or saline. (B) + + fl/+ + Detection of F5-TCR expressing thymocytes in F5P P LckCreP PElavl1P Pand F5P P + fl/fl LckCreP PElavl1P Pmice injected with variable doses of the NP68 peptide. (C) Response of HuR-deficient thymocytes to dexamethasone induced . Thymocytes were cultured in vitro and apoptosis was induced with 100nM dexamethasone. Cells were collected at 0, 4, 16, 24 hours post stimulation. + fl/fl LckCreP PElavl1P Pthymocytes (filled histogram) appear to be as susceptible as control thymocytes (solid line) to dexamethasone induced apoptosis as shown by Annexin V/Propidium iodide staining and flow cytometric analysis. The line + + graphs show both the percentages of dead and apoptotic CD4P PCD8P P cells. Results shown are representative of two independent experiments with n=3 mice/group. (D) qRT-PCR detection of pro-apoptotic mRNAs in + fl/fl LckCreP PElavl1P Pthymocytes. Data (+SD) are fold changes relative to control values (White bar-Ref) and (*) p<0.05. Papadaki et al., 2009 Supplementary

Supplemental Figure 6. Expression of known HuR-targets in activated HuR-deficient thymocytes. Microarray analysis of thymocyte mRNAs previously reported to interact with HuR. Shown are normalized relative + +/+ fl/fl expression values from control LckCreP P Elavl1P P (Ɣ) and Elavl1P P (ż) as well + fl/fl as test LckCreP P Elavl1P P (Ɣ) thymocyte cultures stimulated with PMA/ionomycin for 0,4 and 12 hrs. Papadaki et al., 2009-Supplementary @ Supplementary Table 1a. List of Downregulated in HuR-deficient thymocytes before and after mitogenic stimulationP O Hrs En Gene Name GeneIDP Description Score 1* FC** Score 2* FC** Bbc3 170770 Bcl-2 Binding Component 3 -3,601 1,575 -5,283 1,808 Cacna1e 12290 Voltage-Dependent R-Type Calcium Channel Alpha-1e Subunit -2,734 1,883 -6,186 2,148 Camk2n1 66259 Calcium/Calmodulin-Dependent II Inhibitor -4,542 1,419 -7,791 1,891 Col15a1 12819 Procollagen, Type XV -4,023 1,915 -2,627 2,916 Cyp2j6 74519 Cytochrome P450 2j5 -5,336 1,386 -5,023 1,499 D130020L05Rik 68734 Riken Cdna D130020l05 Gene -3,408 1,409 -2,784 1,206 D15Wsu75e 28075 D15wsu75e Protein -3,235 1,405 -4,176 1,439 Doc2g 60425 Double C2, Gamma -3,183 7,126 -5,934 4,116 Flt4 14257 Vascular Endothelial Growth Factor 3 Precursor -4,063 1,450 -4,644 1,604 Gpr161 240888 G Protein-Coupled Receptor 161 -5,153 1,755 -2,957 1,796 Gzmm 50701 Complement Precursor -2,626 1,391 -3,977 1,227 Lpin1 14245 Lipin 1 (Fatty Liver Dystrophy Protein) -3,779 1,346 -3,655 1,374 Lta 16992 Lymphotoxin-Alpha Precursor (Lt-Alpha) -3,646 2,087 -3,095 1,776 Med25 75613 Mediator Of RNA Polymerase II , Subunit 25 Homolog -2,934 1,147 -2,884 1,152 Oxa1L 69089 Oxidase Assembly 1-Like -3,799 1,420 -4,003 1,379 Phlpp 98432 Ph Domain And Leucine Rich Repeat Protein Prosphatase -3,042 1,368 -2,689 1,628 Ppfibp2 19024 Protein Tyrosine , F Interacting Protein, Binding Protein 2 -5,098 1,625 -3,010 1,346 Q8BZ64-2 654470 Tect1_Mouse Isoform 2 Of Q8bz64 -2,626 1,290 -3,129 1,365 Rtel1 269400 Regulator Of Telomere Elongation Helicase 1 -4,273 1,343 -3,080 1,299 Shf 435684 Src Homology 2 Domain Containing F -4,957 1,427 -10,059 1,449 Slc13a1 55961 Solute Carrier Family 13, Member 1 -3,401 1,913 -3,309 2,114 Ssrp1 20833 Structure-Specific Recognition Protein 1 -2,842 1,287 -3,167 1,182 Stx5a 56389 Syntaxin 5a -3,021 1,350 -4,915 1,359 Tfpi 21788 Tissue Factor Pathway Inhibitor Precursor -9,815 2,178 -10,552 1,757 Tnfrsf25 85030 Receptor Superfamily, Member 25 -3,054 2,133 -3,592 1,886 Unc13a 382018 Unc-13 Homolog A (C. Elegans) -3,057 1,769 -2,710 1,354 4 Hrs Gene Name Gene ID Description Score 1 FC Score 2 FC 1500011H22Rik 68948 Riken Cdna 1500011h22 Gene -7,065 1,403 -2,650 1,292 1700003H04Rik 384775 RIKEN Cdna 1700003H04 GENE -3,359 2,470 -3,232 2,607 4930404H21Rik 73808 RIKEN Cdna 4930404H21 GENE -3,515 1,541 -3,854 1,569 4930579E17Rik 75847 Riken Cdna 4930579e17 Gene -8,278 1,965 -5,110 2,011 4933431D05Rik 381714 RIKEN Cdna 4933431D05 GENE -3,344 1,787 -5,589 2,328 5730557B15Rik 67434 RIKEN Cdna 5730557B15 GENE -3,613 2,324 -3,151 1,992 9430057O19Rik 231093 RIKEN Cdna 9430057O19 GENE -3,344 1,787 -6,657 2,173 Papadaki et al., 2009-Supplementary A830093I24Rik 207921 RIKEN Cdna A830093I24 GENE -3,313 1,319 -11,589 1,839 Abca4 11304 Atp-Binding Cassette, Sub-Family A, Member 4 -5,152 1,651 -2,981 1,289 Atf7ip2 75329 Activating 7 Interacting Protein 2 -9,664 1,292 -3,818 1,175 Camsap1 227634 Calmodulin Regulated Spectrin-Associated Protein 1 -4,479 1,405 -4,112 1,309 Cenpf 108000 Protein F -3,197 1,408 -8,049 1,539 Clec2h 94071 C-Type Lectin Domain Family 2, Member H -3,169 1,908 -2,822 1,484 Cog3 338337 Component Of Oligomeric Golgi Complex 3 -3,223 2,172 -2,639 1,455 Cramp1l 57354 Crm, Cramped-Like -2,699 1,601 -3,110 1,365 Cysltr2 70086 Cysteinyl Leukotriene Receptor 2 -4,376 2,131 -4,768 1,887 Dak 225913 Dihydroxyacetone Kinase 2 Homolog -3,971 1,272 -3,181 1,387 EG639426 639426 Predicted Gene, EG639426 -3,827 1,829 -2,606 1,733 Fbxl22 74165 F-Box And Leucine-Rich Repeat Protein 22 -2,720 2,593 -2,860 2,412 Fndc6 213208 Fibronectin Type Iii Domain Containing 6 -2,701 1,633 -3,344 1,559 Gdpd5 233552 Glycerophosphodiester Domain Containing 5 -3,645 2,119 -2,691 2,510 Glcci1 1E+08 Glucocorticoid Induced Transcript 1; Testhymin -3,368 1,557 -3,067 1,479 Gng2 170772 Guanine Nucleotide Binding Protein (G Protein), Gamma 2 Subunit -2,644 2,075 -3,441 2,511 Gnpda2 14702 Glucosamine-6-Phosphate Deaminase 2 -2,986 1,530 -5,103 1,367 Guf1 67980 Guf1 Gtpase Homolog (S. Cerevisiae) -5,402 2,079 -3,737 2,035 Igf2bp3 231279 -Like Growth Factor 2 Mrna Binding Protein 3 -3,816 1,747 -2,662 1,384 Inpp1 140488 Inositol Polyphosphate 1-Phosphatase (Ec 3.1.3.57) (Ippase) (Ipp). -2,711 1,980 -3,175 1,242 Klrb1c 16329 Killer Cell Lectin-Like Receptor Subfamily B Member 1c -3,249 1,925 -2,645 1,894 Lbh 17057 Limb-Bud And Heart -3,461 1,591 -3,135 2,150 Mizf 77889 Mbd2 (Methyl-Cpg-Binding Protein)-Interacting Protein -3,859 1,323 -3,169 1,334 Mras 102423 Ras-Related Protein M-Ras -3,055 2,018 -2,726 2,463 Ms4a6b 17532 Membrane-Spanning 4-Domains, Subfamily A, Member 6b -3,666 3,243 -2,686 2,878 Nmt1 69774 Peptide N- Myristoyltransferase 1 -2,839 1,677 -5,092 1,339 Olah 18107 Oleoyl-Acp -7,171 1,484 -3,323 1,332 Peci 99035 Peroxisomal 3,2-Trans-Enoyl-Coa -3,804 1,449 -2,940 1,347 Pgls 23986 6-Phosphogluconolactonase -2,859 2,316 -3,141 1,839 Ppp1r16b 1E+08 1 Regulatory Inhibitor Subunit 16b -2,852 2,373 -2,976 1,965 Pygb 228852 Brain Glycogen -5,238 1,395 -9,987 1,691 Rbm5 110078 Rna Binding Motif Protein 5 -3,059 1,654 -3,874 1,464 Rcc2 83486 Regulator Of Chromosome Condensation 2 -3,214 1,372 -5,820 1,331 Rnpep 108911 B -2,788 1,627 -3,361 1,820 Sharpin 215615 Shank-Associated Rh Domain Interacting Protein -4,095 1,397 -2,855 1,180 Sorbs1 106025 Sorbin And Sh3 Domain Containing 1 -2,747 1,201 -3,050 1,284 Tfpi 20411 Tissue Factor Pathway Inhibitor Precursor -5,407 1,756 -3,875 2,378 Tgm6 241636 6 -3,180 1,776 -2,773 1,547 Papadaki et al., 2009-Supplementary Tmem43 74122 43 -4,011 1,539 -2,637 1,164 Tmem63c 217733 Transmembrane Protein 63c -4,837 1,582 -3,295 1,930 Toe1 68276 Target Of Egr1, Member 1 (Nuclear) -2,690 1,888 -4,692 2,165 Tssc1 380752 Tumor Suppressing Subtransferable Candidate 1 -2,961 1,720 -4,159 1,923 12 Hrs Gene Name Gene ID Description Score 1 FC Score 2 FC 1300001I01Rik 74148 Riken Cdna 1300001i01 Gene -3,013 1,631 -2,682 1,550 2610041B18Rik 69930 Riken Cdna 2610041b18 Gene -2,958 2,004 -3,226 1,631 A430041B07Rik 328108 Riken Cdna A430041b07 Gene -2,956 1,550 -3,295 1,642 Adam23 23792 A Disintegrin And Metalloprotease Domain 23 -3,244 2,922 -5,369 2,858 AW060207 231571 Expressed Sequence Aw060207 -4,136 1,755 -8,083 1,908 cobl 12808 Cordon-Bleu Protein -4,332 1,343 -3,344 1,481 Cyp4f13 170716 Cytochrome P450, Family 4, Subfamily F, Polypeptide 13 -4,349 1,332 -3,072 1,235 D1Ertd622e 52392 D1ertd622e Protein -4,360 2,014 -2,930 1,365 Dctd 320685 Dcmp Deaminase -3,049 1,738 -5,353 2,133 Ddx52 78394 Dead (Asp-Glu-Ala-Asp) Box Polypeptide 52 -3,384 1,282 -3,479 1,239 Gart 14450 Glycinamide Ribonucleotide Synthetase -2,788 1,194 -2,930 1,238 Gnb1 14688 Guanine Nucleotide Binding Protein, Beta 1 -2,991 1,405 -3,277 1,476 Igsf9 93842 Immunoglobulin Superfamily, Member 9 -5,312 1,324 -3,062 1,113 Nt5m 103850 5',3'-, Mitochondrial -2,892 1,724 -2,694 1,431 Nudt1 17766 7,8-Dihydro-8-Oxoguanine Triphosphatase (8-Oxo-Dgtpase). -4,156 1,909 -2,954 2,203 Paqr6 68957 Progestin And Adipoq Receptor Family Member Vi -2,915 1,448 -2,914 1,424 Phospho1 237928 Phosphatase, Orphan 1 -3,423 2,182 -3,439 3,282 Pias2 17344 Protein Inhibitor Of Activated Stat 2 -4,910 1,337 -3,789 1,402 Rbpms 19663 RNA-Binding Protein With Multiple Splicing (Rbp-Ms) (Hermes). -7,704 3,155 -2,929 1,878 Rcc2 83486 Regulator Of Chromosome Condensation 2 -2,612 1,264 -6,164 1,561 Tfpi 21788 Tissue Factor Pathway Inhibitor Precursor -2,838 2,018 -4,479 3,080 Tmem177 66343 Transmembrane Protein 177 -2,746 2,050 -3,655 1,481 @: See Also Supplemental Methods En: Gene IDs + fl/fl fl/fl + * Statistical score values reflecting the differential in lckCreP P ElavlP Pthymocytes relative to (1) Elavl1P Por (2) lckCreP P thymocytes + fl/fl fl/fl + ** Fold Change (FC) values reflecting the differential gene expression in lckCreP P ElavlP Pthymocytes relative to (1) Elavl1P Por (2) lckCrePP thymocytes Papadaki et al., 2009-Supplementary

@ Supplementary Table 1b. List of Upregulated Genes in HuR- deficient thymocytes before and after mitogenic stimulationP O Hrs En Gene Name Gene IDP P Description Score 1* FC** Score 2* FC** 1110015M06Rik 68511 RIKEN CDNA 1110015m06 Gene 2,765 1,738 3,493 1,818 4931414P19Rik 74359 RIKEN CDNA 4931414P19 Gene 2,962 1,262 6,084 1,538 5830477G23Rik RIKEN CDNA 5830477g23 Gene 3,967 1,540 3,766 1,421 Agl 77559 Amylo-1,6-Glucosidase, 4-Alpha-Glucanotransferase 6,482 1,628 4,657 1,390 Alx4 11695 Protein Aristaless-Like 4 3,452 1,390 2,863 1,321 Asb8 78541 Ankyrin Repeat And Socs Box-Containing Protein 8 4,315 1,234 3,578 1,243 Btbd16 330660 Btb (Poz) Domain Containing 16 5,101 2,986 6,786 1,528 D15Ertd621e 210998 Hypothetical Protein 2,759 1,190 3,248 1,235 D230004J03Rik 194388 Riken Cdna D230004j03 Gene 3,355 1,170 3,985 1,465 Dync1li2 234663 Dynein, Cytoplasmic 1 Light Intermediate Chain 2 2,891 1,253 2,920 1,399 Heca 380629 Headcase Homolog (Drosophila) 5,644 1,903 4,855 2,228 Itga1 109700 Integrin Alpha 1 3,095 2,131 4,012 2,200 Lactb 80907 Beta Lactamase-Like Protein 4,869 1,249 9,215 1,421 Mad2l2 71890 Mad2 Mitotic Arrest Deficient-Like 2 (Yeast) 2,954 1,233 3,178 1,240 Mrps12 24030 28s Ribosomal Protein S12, Mitochondrial Precursor (Mpr-S12) 3,012 1,330 2,905 1,299 Nup 160 59015 160 2,851 1,393 3,652 1,701 Olfr94 258219 Olfactory Receptor 94 2,947 1,851 2,720 1,778 Pdss2 71365 Prenyl (Solanesyl) Diphosphate Synthase, Subunit 2 2,815 1,456 3,849 1,748 Polr3a 218832 Polymerase (Rna) Iii (Dna Directed) Polypeptide A 2,979 1,712 2,702 1,785 Q9CQV7-2 67713 Tim14_Mouse Isoform 2 Of Q9cqv7 4,296 1,169 3,732 1,237 Spryd3 223918 Spry Domain Containing 3 5,734 1,437 3,329 1,347 Tas2r10 667992 Candidate Taste Receptor Trb2 3,962 1,628 4,298 1,524 Txndc8 67402 Thioredoxin Domain Containing 8 3,381 2,225 3,080 2,191 4 Hrs En Gene Name Gene IDP P Description Score 1* FC** Score 2* FC** 0610012G03Rik 106264 RIKEN Cdna 0610012G03 Gene 2,940 1,230 4,075 1,416 1110001J03Rik 66117 Riken Cdna 1110001j03 Gene 4,064 1,455 4,737 1,429 1110007M04Rik 68493 Riken Cdna 1110007m04 Gene 3,772 1,266 4,727 1,278 2610511M17Rik 74206 Riken Cdna 2610511m17 Gene 2,723 1,383 2,612 1,226 2810410M20Rik 66310 Riken Cdna 2810410m20 Gene 2,836 1,364 2,871 1,158 4631427C17Rik 74340 Riken Cdna 4631427c17 Gene 3,107 1,595 3,336 1,520 A230046K03Rik 319277 Riken Cdna A230046k03 Gene 3,718 1,431 3,782 1,629 Acpt 546967 , Testicular 4,347 1,593 7,726 1,577 Akt3 23797 Protein Kinase Akt-3; (, Gamma) (Pkb Gamma) 3,300 2,150 3,050 1,935 Papadaki et al., 2009-Supplementary Amot 27494 Angiomotin 2,823 1,591 3,100 1,903 Aof1 218214 Amine Oxidase, Flavin Containing 1 2,943 1,437 4,200 1,420 BC038156 235132 CDNA Sequence Bc038156 2,736 1,163 4,453 1,293 Bmyc 107771 Brain Expressed Myelocytomatosis 12,322 1,497 2,735 1,231 Bri3bp 76809 Bri3 Binding Protein 3,562 1,644 4,129 1,560 Camta1 100072 Calmodulin Binding Transcription Activator 1 2,701 1,543 6,652 1,938 Canx 12330 Calnexin Precursor 3,031 1,259 5,123 1,387 Carm1 59035 Coactivator-Associated Arginine Methyltransferase 1 3,104 1,704 3,108 1,716 Cd3e 12501 T-Cell Surface Glycoprotein Cd3 Epsilon Chain Precursor 3,301 1,431 7,781 1,678 Cdc25a 12530 M-Phase Inducer Phosphatase 1/(Dual Specificity Phosphatase Cdc25a) 5,157 1,399 2,753 1,327 Cspg5 29873 Chondroitin Sulfate Proteoglycan 5; Neuroglycan C 4,181 2,188 2,895 1,627 Cyb5r1 72017 Cytochrome B5 Reductase 1 2,871 1,845 5,007 2,293 EG432466 432466 Predicted Gene, EG432466 3,925 1,833 3,712 1,362 Fndc3b 72007 Fibronectin Type Iii Domain Containing 3b 2,820 1,614 3,176 1,869 Fras1 231470 Fraser Syndrome 1 Homolog (Human) 2,640 1,624 3,390 1,489 Gpr172b 52710 G Protein-Coupled Receptor 172b 5,181 2,103 3,127 1,198 Mapk12 29857 Mitogen-Activated Protein Kinase 12 (Map Kinase P38 Gamma). 4,873 2,297 3,426 1,745 624424 Predicted Gene, OTTMUSG00000007747 4,721 1,227 3,239 1,149 P4ha3 320452 Proline 4-Hydroxylase 2,632 1,524 4,299 2,533 Ppp4c 56420 Protein Phosphatase 4, Catalytic Subunit 4,121 1,336 3,261 1,259 Ptgs1 19224 Prostaglandin G/H Synthase 1 Precursor (Cyclooxygenase -1) (Cox-1) 3,451 1,863 5,550 2,591 Rab13 68328 Rab13, Member Ras Oncogene Family 5,223 2,155 3,330 1,625 Rgs17 56533 Regulator Of G-Protein Signaling 17 5,580 2,747 5,428 1,763 Rsad2 58185 Radical S-Adenosyl Methionine Domain Containing 2 4,704 2,098 7,271 2,362 Rufy2 70432 Run And Fyve Domain-Containing 2 4,245 2,236 3,785 1,937 Sdc4 20971 Syndecan-4 Precursor (Ryudocan Core Protein) 4,070 2,174 3,283 1,776 Sfrs2 20382 Splicing Factor, Arginine/Serine-Rich 2 (Splicing Factor Sc35) (Sc-35) 4,141 1,720 4,792 1,951 Sh2b3 16923 Sh2b Adaptor Protein 3 3,035 1,612 2,610 1,474 Slc9a8 77031 Solute Carrier Family 9 (Sodium/Hydrogen Exchanger), Member 8 3,698 1,395 3,582 1,320 Stc2 20856 Stanniocalcin 2 Precursor (Stc-2) 2,921 2,015 3,901 1,780 Tgm2 21817 Protein-Glutamine Gamma-Glutamyltransferase 2,765 1,736 3,638 1,490 Tspan4 64540 Tetraspanin 4 3,558 1,865 3,917 2,480 Vav1 22324 Vav Proto-Oncogene 3,259 1,186 3,267 1,444 Xkr8 381560 X Kell Blood Group Precursor Related Family Member 8 Homolog 2,933 1,570 5,165 2,042 12 Hrs En Gene Name Gene IDP P Description Score 1* FC** Score 2* FC** 4930461P20RIk 78244 Riken Cdna 4930461p20 Gene 4,150 1,282 2,977 1,083 1110002E23Rik 66119 Riken Cdna 1110002e23 Gene 3,508 1,759 2,734 1,420 Papadaki et al., 2009-Supplementary 1810023F06Rik 217845 Riken Cdna 1810023f06 Gene 3,572 1,880 4,030 1,929 2010107G23Rik 69894 Riken Cdna 2010107g23 Gene 2,673 1,483 4,525 1,436 2410002F23Rik 668661 RIKEN Cdna 2410002F23 Gene 2,743 1,393 2,778 1,249 Aarsl 224805 Alanyl-Trna Synthetase Like 2,644 1,394 5,299 1,501 AI597013 100182 Expressed Sequence Ai597013 4,488 1,886 2,619 1,700 Ak3l1 11639 Adenylate Kinase 3 Alpha-Like 1 2,772 1,524 3,234 1,981 Atg3 67841 Autophagy-Related 3 (Yeast) 3,277 1,754 6,937 1,448 Atp1b1 11931 Sodium/Potassium-Transporting Atpase Beta-1 Chain 5,632 1,345 10,279 1,592 Brd1 223770 Bromodomain Containing 1 3,380 1,348 4,610 1,411 C4bp 12269 C4b-Binding Protein Precursor (C4bp). [Source:Swissprot;Acc:P08607] 2,615 1,534 3,389 1,471 Camta1 100072 Calmodulin Binding Transcription Activator 1 2,867 1,507 3,696 1,789 Casp8ap2 26885 Caspase 8 Associated Protein 2 2,911 1,219 4,379 1,274 Cbx8 30951 Chromobox Protein Homolog 8 (Polycomb 3 Homolog) (Pc3) (Mpc3) 3,042 1,358 4,042 1,358 Ccdc7 74703 Coiled-Coil Domain Containing 7 2,697 1,955 3,152 2,002 Cd2 12481 T-Cell Surface Antigen CD2 Precursor (Lfa-2) (Lfa-3 Receptor) 4,133 1,591 7,682 2,212 Cdc25b 12531 M-Phase Inducer Phosphatase 2 (Dual Specificity Phosphatase Cdc25b) 3,256 1,784 3,207 1,691 Cmtm7 102545 Cklf-Like Marvel Transmembrane Domain Containing 8 3,473 1,833 2,847 1,453 Coro7 78885 Coronin 7 3,182 1,266 3,688 1,433 Cuta 67675 Divalent Cation Tolerant Protein Cuta 6,204 1,342 4,381 1,317 Cwf19l2 244672 Cwf19-Like 2, Cell Cycle Control (S. Pombe) 2,625 1,697 2,960 1,620 Cxcl16 66102 Cxc Chemokine Ligand 16 5,011 1,770 4,261 1,278 Dntt 21673 Deoxynucleotidyltransferase, Terminal 4,610 5,306 4,020 3,419 Dpm2 13481 Dolichol-Phosphate (Beta-D) Mannosyltransferase 2 3,410 1,220 2,944 1,278 Dpp4 13482 Iv (T-Cell Activation Antigen CD26) 3,327 2,210 4,509 2,157 Edem2 108687 Er Degradation Enhancer, Alpha-Like 2 2,708 1,279 3,143 1,304 Ergic3 66366 Ergic And Golgi 3 3,039 1,274 3,372 1,430 Fibp 58249 Acidic Intracellular Binding Protein 2,878 1,348 6,165 1,205 Galnt2 108148 N-Acetylgalactosaminyltransferase 2 3,249 1,615 2,970 1,505 Gbp3 55932 Guanylate Nucleotide Binding Protein 3] 2,787 1,507 2,866 1,743 il24 13433 Interleukin 24 4,283 1,515 3,339 1,593 Itfg2 101142 Integrin Alpha Fg-Gap Repeat Containing 2 4,965 1,588 4,601 1,459 Jarid2 16468 Jumonji, At Rich Interactive Domain 2 2,726 1,541 3,880 1,725 Lgals9 16859 Galectin-9. [Source:Swissprot;Acc:O08573] 3,350 1,399 3,502 1,460 Mtmr12 268783 Vmyotubularin Related Protein 12 3,512 1,200 3,548 1,211 Mylc2pl 59310 Myosin Light Chain 2, Precursor Lymphocyte-Specific 2,989 1,972 3,746 2,218 Nfkbie 18037 Nf-Kappab Inhibitor Epsilon (Nf-Kappa-Bie) (I-Kappa-B-Epsilon) 6,327 1,385 3,401 1,446 Ngfrap1 12070 Nerve Growth Factor Receptor (Tnfrsf16) Associated Protein 1 2,897 1,203 3,489 1,310 Nup62 18226 Glycoprotein P62 (62 Kda Nucleoporin) 4,789 1,266 3,522 1,111 Papadaki et al., 2009-Supplementary Otop3 69602 Otopetrin 3 4,082 1,700 5,585 1,740 Otx2 18424 Homeobox Protein Otx2 2,796 1,697 2,786 1,722 Pdia4 12304 Protein Disulfide Isomerase Associated 4 3,613 1,486 4,083 1,703 Pdzk1ip1 67182 Pdzk1 Interacting Protein 1 3,264 1,488 3,188 1,271 Phc2 54383 Polyhomeotic-Like 2; Polyhomeotic 2 Protein 2,812 1,407 3,319 1,453 Polg2 50776 Dna Polymerase Gamma Subunit 2, Mitochondrial Precursor 5,243 2,717 5,116 2,506 Pp11r 19011 Placental Protein 11 Related 4,892 2,315 5,822 2,231 Ppp1r16b 228852 Regulatory Inhibitor Subunit 16b 2,807 1,245 4,785 1,345 Pycard 66824 Pyd And Card Domain Containing 2,636 1,674 2,688 1,761 R3hdm2 71750 R3h Domain Containing 2 5,284 1,538 2,790 1,140 Rapgef3 223864 Rap Guanine Nucleotide Exchange Factor (Gef) 3 4,831 2,645 3,031 2,074 Rasgrp2 19395 Ras, Guanyl Releasing Protein 2 4,787 2,036 5,795 2,226 Rlf 109263 Rearranged L- Fusion Sequence 4,043 1,452 2,767 1,284 Runx1 12394 Runt-Related Transcription Factor 1 (Oncogene Aml-1) 2,676 1,382 9,946 1,527 Scin 20259 Adseverin (Scinderin) (Gelsolin-Like Protein) 5,712 2,102 3,387 1,601 Sla 20491 Src-Like Adaptor; Src-Like Adapter Protein; Src-Like Adaptor Protein. 3,536 1,939 3,049 1,711 Sqstm1 18412 Sequestosome 1; Induced 7,444 1,376 4,718 1,346 Srebf2 20788 Sterol Regulatory Element Binding Factor 2 5,490 1,429 4,556 1,542 Stambpl1 76630 Stam Binding Protein Like 1 2,609 1,314 3,085 1,372 Stat3 20848 Signal Transducer And Activator Of Transcription 3 3,411 1,632 3,769 1,598 Stk38 106504 Serine/Threonine Kinase 38 5,269 1,920 6,047 2,191 Sult1c1 69083 Sulfotransferase Family, Cytosolic, 1c, Member 1 4,091 1,992 3,336 1,551 Syt13 80976 Synaptotagmin 13 4,661 1,465 3,748 1,668 Tdp1 104884 Tyrosyl-Dna Phosphodiesterase 1 4,728 1,412 2,768 1,401 Thap3 69876 Thap Domain Containing, Apoptosis Associated Protein 3 4,058 1,371 2,893 1,350 Tnf 21926 Tumor Necrosis Factor Precursor (Tnf-Alpha) 4,124 2,293 3,484 2,092 Trp53inp1 60599 Transformation Related Protein 53 Inducible 1 3,258 1,482 2,870 1,666 Tspan32 27027 Tetraspanin 32 4,080 1,691 3,158 2,016 Vash1 238328 Vasohibin 1 2,687 1,715 3,002 1,637 Wnk2 75607 Wnk Deficient Protein Kinase 2 3,216 1,339 4,530 1,846 Xdh 22436 Xanthine Dehydrogenase/Oxidase 2,804 1,339 8,521 1,717 XR_003475.1 Similar To Ribosomal Protein L18a (Loc625760), Mrna 4,836 1,285 3,314 1,242 @: See Also Supplemental Methods En: Entrez Gene IDs + fl/fl fl/fl + * Statistical score values reflecting the differential gene expression in lckCreP P ElavlP Pthymocytes relative to (1) Elavl1P Por (2) lckCreP P thymocytes + fl/fl fl/fl + ** Fold Change (FC) values reflecting the differential gene expression in lckCreP P ElavlP Pthymocytes relative to (1) Elavl1P Por (2) lckCrePP thymocytes Papadaki et al., 2009-Supplementary

Supplementary Table 2. List of Genes relating to an “Enlarged Thymus” phenotype used as a training set for the ENDEAVOUR software application

MGI ID Gene Description MGI:99607 Abca1 ATP-binding cassette, sub-family A (ABC1), member 1 MGI:87916 Ada adenosine deaminase MGI:105043 Ahr aryl-hydrocarbon receptor MGI:87986 Akt1 thymoma viral proto-oncogene 1 MGI:107928 Aldh1a2 aldehyde dehydrogenase family 1, subfamily A2 MGI:88024 Ank1 ankyrin 1, erythroid MGI:88031 Anxa7 annexin A7 MGI:88039 Apc adenomatosis polyposis coli MGI:88057 Apoe apolipoprotein E MGI:88064 Ar MGI:107202 Atm ataxia telangiectasia mutated homolog (human) MGI:88127 B2m beta-2 microglobulin MGI:1929913 Bcl11b B-cell leukemia/lymphoma 11B MGI:88138 Bcl2 B-cell leukemia/lymphoma 2 MGI:1197519 Bcl2l11 BCL2-like 11 (apoptosis facilitator) MGI:107187 Bcl6 B-cell leukemia/lymphoma 6 MGI:1097714 Bhlhb2 basic helix-loop-helix domain containing, class B2 MGI:1206591 Bik BCL2-interacting killer MGI:2176433 Bmf BCL2 modifying factor MGI:103301 Cacnb4 calcium channel, voltage-dependent, beta 4 subunit MGI:88279 Cbl Casitas B-lineage lymphoma MGI:88313 Ccnd1 cyclin D1 MGI:88334 Cd247 CD247 antigen MGI:1330281 Cd2ap CD2-associated protein MGI:88335 Cd4 CD4 antigen MGI:88337 Cd40lg CD40 ligand MGI:104565 Cdkn1b cyclin-dependent kinase inhibitor 1B MGI:105388 Cdkn2c cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) MGI:88388 Cftr cystic fibrosis transmembrane conductance regulator homolog MGI:1313268 Chrd chordin MGI:88467 Col1a1 collagen, type I, alpha 1 MGI:104686 Crkl v-crk sarcoma virus CT10 oncogene homolog (avian)-like MGI:1339753 Csf1 colony stimulating factor 1 (macrophage) Papadaki et al., 2009-Supplementary MGI:88537 Csk c-src tyrosine kinase MGI:88556 Ctla4 cytotoxic T-lymphocyte-associated protein 4 MGI:88564 Ctsl L MGI:88578 Cycs cytochrome c, somatic MGI:1921506 Cyld cylindromatosis (turban tumor syndrome) MGI:88588 Cyp1a1 cytochrome P450, family 1, subfamily a, polypeptide 1 MGI:88589 Cyp1a2 cytochrome P450, family 1, subfamily a, polypeptide 2 MGI:2177632 Cys1 cystin 1 MGI:94872 Dcn decorin MGI:1329019 Dnase2a II alpha MGI:1261819 Dnmt3b DNA methyltransferase 3B MGI:893587 Dok1 docking protein 1 MGI:1332623 Dok2 docking protein 2 MGI:101941 transcription factor 1 MGI:1096341 E2F transcription factor 2 MGI:1101357 Ece1 endothelin converting 1 MGI:105923 Ednra endothelin receptor type A MGI:1928377 Elf4 E74-like factor 4 (ets domain transcription factor) MGI:1933403 Erap1 endoplasmic reticulum aminopeptidase 1 MGI:1921405 Errfi1 ERBB receptor feedback inhibitor 1 MGI:95455 Ets1 E26 avian leukemia oncogene 1, 5' domain MGI:99960 Ewsr1 Ewing sarcoma breakpoint region 1 MGI:109344 Eya1 eyes absent 1 homolog (Drosophila) MGI:95484 Fas Fas (TNF receptor superfamily member 6) MGI:99255 Fasl Fas ligand (TNF superfamily, member 6) MGI:1354695 Fbxw7 F-box and WD-40 domain protein 7 MGI:1099809 Fgf10 fibroblast growth factor 10 MGI:99604 Fgf8 fibroblast growth factor 8 MGI:95523 Fgfr2 fibroblast growth factor receptor 2 MGI:1347464 Foxg1 forkhead box G1 MGI:1891436 Foxp3 forkhead box P3 MGI:1313278 Fzd9 frizzled homolog 9 (Drosophila) MGI:107799 Gadd45a growth arrest and DNA-damage-inducible 45 alpha MGI:95665 Gba glucosidase, beta, acid MGI:95668 Gbx2 gastrulation brain homeobox 2 MGI:95841 Gsc goosecoid homeobox MGI:1353434 Gulo gulonolactone (L-) oxidase MGI:95895 H2-Aa histocompatibility 2, class II antigen A, alpha MGI:103070 H2-Ab1 histocompatibility 2, class II antigen A, beta 1 Papadaki et al., 2009-Supplementary MGI:1931526 Hist1h1c histone cluster 1, H1c MGI:107502 Hist1h1d histone cluster 1, H1d MGI:1931527 Hist1h1e histone cluster 1, H1e MGI:96175 Hoxa3 homeo box A3 MGI:96448 Igh-6 immunoglobulin heavy chain 6 (heavy chain of IgM) MGI:1342540 Ikzf1 IKAROS family zinc finger 1 MGI:96537 Il10 interleukin 10 MGI:96548 Il2 interleukin 2 MGI:96549 Il2ra interleukin 2 receptor, alpha chain MGI:96550 Il2rb interleukin 2 receptor, beta chain MGI:2685460 Gm614 gene model 614, (NCBI) MGI:96551 Il2rg interleukin 2 receptor, gamma chain MGI:105303 Il8rb interleukin 8 receptor, beta MGI:1096873 Irf4 interferon regulatory factor 4 MGI:96395 Irf8 interferon regulatory factor 8 MGI:96606 Itgal integrin alpha L MGI:108427 Insl3 insulin-like 3 MGI:99928 Jak3 Janus kinase 3 MGI:104813 Jarid2 jumonji, AT rich interactive domain 2 MGI:107566 Kifap3 kinesin-associated protein 3 MGI:2148793 Kiss1r KISS1 receptor MGI:96677 Kit kit oncogene MGI:1101771 Kl klotho MGI:96680 Kras v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog MGI:106588 Lag3 lymphocyte-activation gene 3 MGI:96748 Lamp2 lysosomal-associated membrane protein 2 MGI:1342293 Lat linker for activation of T cells MGI:1891468 Lgr4 leucine-rich repeat-containing G protein-coupled receptor 4 MGI:1917780 Lrfn2 leucine rich repeat and fibronectin type III domain containing 2 MGI:1346877 Map3k7 mitogen-activated protein kinase kinase kinase 7 MGI:1346861 Mapk8 mitogen-activated protein kinase 8 MGI:1346862 Mapk9 mitogen-activated protein kinase 9 MGI:96928 Mc2r melanocortin 2receptor MGI:107785 Mesp1 mesoderm posterior 1 MGI:97250 Myc myelocytomatosis oncogene MGI:102463 Nfatc2 nuclear factor of activated T-cells, -dependent 2 MGI:103296 Nfatc3 nuclear factor of activated T-cells, calcineurin-dependent 3 MGI:97350 Nkx2-5 NK2 transcription factor related, locus 5 (Drosophila) MGI:1097712 Npc1 Niemann Pick type C1 Papadaki et al., 2009-Supplementary MGI:1352454 Nr4a1 subfamily 4, group A, member 1 MGI:1352457 Nr4a3 nuclear receptor subfamily 4, group A, member 3 MGI:97487 Pax3 paired box gene 3 MGI:97493 Pax9 paired box gene 9 MGI:103248 Phc1 polyhomeotic-like 1(Drosophila) MGI:1353576 Pik3cg phosphoinositide-3-kinase, catalytic, gamma polypeptide MGI:97615 Plcg1 C, gamma 1 MGI:1858214 Ppm1d protein phosphatase 1D magnesium-dependent, delta isoform MGI:1923810 Prss8 , serine, 8 (prostasin) MGI:109583 Pten phosphatase and tensin homolog MGI:97806 Ptpn2 protein tyrosine phosphatase, non-receptor type 2 MGI:96055 Ptpn6 protein tyrosine phosphatase, non-receptor type 6 MGI:1928376 Ptpn9 protein tyrosine phosphatase, non-receptor type 9 MGI:97810 Ptprc protein tyrosine phosphatase, receptor type, C MGI:1353578 Pttg1 pituitary tumor-transforming 1 MGI:109292 Rad50 RAD50 homolog (S. cerevisiae) MGI:97848 Rag1 recombination activating gene 1 MGI:97849 Rag2 recombination activating gene 2 MGI:97897 Rel reticuloendotheliosis oncogene MGI:103290 Rela v-rel reticuloendotheliosis viral oncogene homolog A (avian) MGI:103289 Relb avian reticuloendotheliosis viral (v-rel) oncogene related B MGI:1098272 Rnasel L (2', 5'-oligoisoadenylate synthetase-dependent) MGI:104856 Rorc RAR-related orphan receptor gamma MGI:99852 Runx1 runt related transcription factor 1 MGI:99829 Runx2 runt related transcription factor 2 MGI:102672 Runx3 runt related transcription factor 3 MGI:98282 Sfpi1 SFFV proviral integration 1 MGI:98284 Sfrs2 splicing factor, arginine/serine-rich 2 (SC-35) MGI:1889342 Sit1 suppression inducing transmembrane adaptor 1 MGI:106203 Skil SKI-like MGI:892001 Slc22a6 solute carrier family 22 (organic anion transporter), member 6 MGI:1926080 Slc25a12 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 MGI:1201674 Smad3 MAD homolog 3 (Drosophila) MGI:1096393 Snai2 snail homolog 2 (Drosophila) MGI:1354910 Socs1 suppressor of signaling 1 MGI:98397 Src Rous sarcoma oncogene MGI:103063 Stat1 signal transducer and activator of transcription 1 MGI:103034 Stat6 signal transducer and activator of transcription 6 MGI:107476 Stim1 stromal interaction molecule 1 Papadaki et al., 2009-Supplementary MGI:2151156 Stim2 stromal interaction molecule 2 MGI:98493 Tbx1 T-box 1 MGI:98510 Tcfe2a transcription factor E2a MGI:98623 Tcrg T-cell receptor gamma chain MGI:98725 Tgfb1 transforming growth factor, beta 1 MGI:104798 Tnf tumor necrosis factor MGI:1341090 Tnfrsf10b tumor necrosis factor receptor superfamily, member 10b MGI:1314891 Tnfrsf11a tumor necrosis factor receptor superfamily, member 11a MGI:1934667 Tnfrsf25 tumor necrosis factor receptor superfamily, member 25 MGI:99908 Tnfrsf8 tumor necrosis factor receptor superfamily, member 8 MGI:1100089 Tnfsf11 tumor necrosis factor (ligand) superfamily, member 11 MGI:102724 Tpp2 II MGI:108072 Traf6 Tnf receptor-associated factor 6 MGI:108072 Traf6 Tnf receptor-associated factor 6 MGI:1328317 Trex1 three prime repair 1 MGI:98834 Trp53 transformation related protein 53 MGI:1920999 Ttc7 tetratricopeptide repeat domain 7 MGI:98872 Twist1 twist gene homolog 1 (Drosophila) MGI:109352 Ung uracil DNA MGI:103076 Vdr MGI:103178 Vegfa vascular endothelial growth factor A MGI:2682003 Whrn whirlin MGI:99180 Zfp36 zinc finger protein 36 MGI:1890508 Zmpste24 zinc metallopeptidase, STE24 homolog (S. cerevisiae)

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