SUPPLEMENTARY INFORMATION. Pharmacological correction of a defect in PPAR-γ signaling ameliorates disease severity in Cftr-deficient mice

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1 SUPPLEMENTARY INFORMATION Pharmacological correction of a defect in PPAR-γ signaling ameliorates disease severity in Cftr-deficient mice Gregory S Harmon 1, Darren S Dumlao 2,3, Damian T Ng 4, Kim E Barrett 1, Edward A Dennis 2,3, Hui Dong 1 and Christopher K Glass 1,4 1 Department of Medicine, 2 Department of Chemistry and Biochemistry, 3 Department of Pharmacology and 4 Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA. Correspondence should be addressed to G.S. (gharmon@ucsd.edu) or C.K.G. (ckg@ucsd.edu). SUPPLEMENTARY METHODS Mice. Mice carrying the loxp-targeted Pparg were generated as described 1. The mice were crossed with Vil1-Cre transgenic mice, generated as described 2 (Jackson Laboratory), and backcrossed eight generations to C57BL6/J mice (Jackson Laboratory, Stock ). Intestinal epithelial cell isolation. Colonic epithelial cells were harvested from sibling female wild-type or Cftr -/- and Pparg f/f or Pparg IEC-/- mice using a modification of the method as described 3. Briefly, intestinal tissue was dissected, washed in phosphate buffered saline (PBS) and incubated in 2 mm EDTA and 1 mm EGTA in Hanks balanced salt solution without calcium or magnesium for 12 min at 37 C. The supernatant was discarded and the remaining mucosa vortexed in ice cold PBS. The suspension was centrifuged (75 x g for 5 min at 4 C) to isolate intact crypts, the supernatant discarded, and the pellet retained at -80 C for RNA and protein analysis.

2 Transcriptome analysis. Two Sentrix Mouse-Ref8 v1.1 Expression Beadchips (Illumina) were used to identify differentially expressed genes from 16,075 probes between littermate wild-type and Cftr -/- mice treated with or without rosiglitazone as previously described 4. Briefly, 1 µg RNA isolated as described above from each sample was amplified to cdna, transcribed to crna, biotin labeled, hybridized and detected with streptavidin-cy3 following the manufacturer s protocol. BeadChips were scanned with Illumina BeadArray Reader and analyzed as previously described 5. Data were normalized and analyzed with GENESPRING (Silicon Genetics). GeneOntology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) pathway analyses were performed using the DAVID bioinformatics resource as described 6. Genes down- or upregulated in knock-out mice were identified using a false discovery rate of 0.05 and a maximal P value of 0.10 (unpaired, two-tailed t-test) and connected to biological process annotations provided by the GO Consortium 7. Cell culture. The human colonic epithelial cell line, T84, was cultured in Dulbecco s modified Eagle s medium/hams F-12 medium (1:1) supplemented with 5% newborn calf serum (HyClone). COS-7 cells (ATCC) were maintained in Dulbecco s modified Eagle s medium supplemented with 10% fetal calf serum (Hyclone). For Ussing chamber studies, T84 cells were seeded on 12-mm Millicell-HA inserts (Millipore) and cultured for 21 days to achieve transepithelial resistance of >1200 Ω cm 2 as measured by an epithelial voltohmmeter (EVOM, World Precision Instruments) prior to experiments. Colonic epithelial ion transport. Segments of mid-distal colon were stripped of muscle layers and mounted on Ussing chamber inserts with a window area of 0.07 cm 2 as described previously 8. Tissues were bathed in oxygenated Ringer s solution at 37 C containing (in mm) 140 Na, 5.2 K, 1.2 Ca 2, 0.8 Mg 2, 120 Cl -, 25 HCO - 3, 2.4 H 2 PO - 4, 0.4 HPO 2-4, and 10 glucose. The tissues were voltage-clamped to zero potential difference and short-circuit current recorded using a DVC-1000 Dual voltage-clamp (World Precision Instruments). Results were normalized and expressed as ΔIsc

3 (µa/cm 2 ). Chloride-free Ringer s solution contained (in mm) 145 Na, 5.2 K, 1.2 Ca 2, 1.2 Mg 2, 2.4 SO 2-4, 2.8 PO 3-4, 25 HCO - 3, isethionate and 10 glucose. Apical chloride-free solution was substituted to increase the driving force for chloride transport and provide a measurable ΔIsc in Cftr -/- colon following the addition of carbachol. Shortcircuit current was recorded in response to application of forskolin (10 µm) at the mucosal and serosal surface or carbachol (100 µm) at the serosal surface. T84 cells were grown as monolayers on Millicell-HA supports and mounted in Ussing chambers with a window of 0.6 cm 2 as described 9. For CFTR inhibitor studies, the basolateral membrane was permeabilized with 100 µm amphotericin B (Sigma) followed by treatment with 10 µm CFTR inh -172 (Sigma) at the basolateral surface. The buffer was changed to standard Ringer s on the basolateral side and chloride-free Ringer s on the apical side, and the response to forskolin or carbachol recorded. Transient transfection and reporter studies. COS-7 cells were transfected with 0.1 µg of the luciferase reporter plasmid and 0.1 µg of the PPARγ expression plasmid or empty vector with FuGENE 6 Transfection Reagent (Roche). A β-galactosidase expression vector was co-transfected as an internal control. The reporter construct was ptal (Clonetech). Enhancers containing bp sequence were cloned from C57Bl/6 genomic DNA to the reporter construct using Phusion High-Fidelty DNA Polymerase (Finnzymes). Cells were treated with Ro (1 µm) as indicated. Luciferase reporter gene activity was measure after 24 hr Ro treatment and performed as previously described 10. Lipidomic analysis. The sample preparation, liquid chromatography mass spectrometry, and gas chromatography mass spectrometry were conducted as previously described Adaptations to the LC/MS/MS method include using the scheduled MRM mode (Analyst 1.5 software, Applied Biosystems) to increase the total MRM transitions monitored to 174. Multiquant 1.1 software (Applied Biosystems) was used in the quantitation of each metabolite. Briefly, right colon epithelial cells from six wild-type and five Cftr -/- were harvested via scrapping, weighed and immediately frozen. Cells were suspended in solution containing 800 µl of H 2 O, 200 µl of a deuterated mix of

4 internal fatty acid and eicosanoid standards in methanol. Cells were lysed using a sonicator probe. 80 µl was removed for liquid liquid extraction and fatty acid analysis by gas chromatography mass spectrometry (GC/MS). 920 µl was subjected to solid phase extraction. All extracted samples were stored at -80 C until analyzed via mass spectrometry. Extracted eicosanoid samples were dried under vacuum centrifugation before resuspension in 100 µl of solvent A (H 2 O-acetonitrile-acetic acid (70:30:0.02; v/v/v)). An aliquot of 40 µl was injected onto a reverse-phase HPLC system linked in series with an Applied Biosystems Q-Trap Fatty acids were analyzed using an Agilent Technologies GC 6890N linked with a 5975 imsd. SUPPLEMENTARY REFERENCES 1. Hevener, A.L., et al. Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones. J Clin Invest 117, (2007). 2. Madison, B.B., et al. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J Biol Chem 277, (2002). 3. Rogler, G., et al. Establishment of long-term primary cultures of human small and large intestinal epithelial cells. Lab Invest 78, (1998). 4. Ogawa, S., et al. Molecular determinants of crosstalk between nuclear receptors and toll-like receptors. Cell 122, (2005). 5. Sasik, R., Calvo, E. & Corbeil, J. Statistical analysis of high-density oligonucleotide arrays: a multiplicative noise model. Bioinformatics 18, (2002). 6. Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, (2009). 7. Creating the gene ontology resource: design and implementation. Genome Res 11, (2001). 8. McCole, D.F., Rogler, G., Varki, N. & Barrett, K.E. Epidermal growth factor partially restores colonic ion transport responses in mouse models of chronic colitis. Gastroenterology 129, (2005). 9. Chappell, A.E., et al. Hydrogen peroxide inhibits Ca2-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins. FASEB J 22, (2008). 10. Pascual, G., et al. A SUMOylation-dependent pathway mediates transrepression of inflammatory response genes by PPAR-gamma. Nature 437, (2005).

5 11. Blaho, V.A., Buczynski, M.W., Brown, C.R. & Dennis, E.A. Lipidomic analysis of dynamic eicosanoid responses during the induction and resolution of Lyme arthritis. J Biol Chem 284, (2009). 12. Zarini, S., Gijon, M.A., Folco, G. & Murphy, R.C. Effect of arachidonic acid reacylation on leukotriene biosynthesis in human neutrophils stimulated with granulocyte-macrophage colony-stimulating factor and formyl-methionyl-leucylphenylalanine. J Biol Chem 281, (2006). 13. Quehenberger, O., Armando, A., Dumlao, D., Stephens, D.L. & Dennis, E.A. Lipidomics analysis of essential fatty acids in macrophages. Prostaglandins Leukot Essent Fatty Acids 79, (2008).

6 Supplementary Figure Legends Supplementary Figure 1. Effect of PPARγ activation on gene expression in mice. (a) GeneOntology (GO) terms significantly enriched in genes down-regulated and upregulated in Cftr -/- colonic epithelial cells, indicating p values and Benjamini correction for multiple hypothesis testing. (b) Effect of Ro treatment on expression of 388 genes down-regulated and 328 genes up-regulated in Cftr -/- colonic epithelial cells compared to control cells. Supplementary Figure 2. Additional PPARγ-dependent target genes in Cftr- and Pparg-deficient mice. (a) Q-PCR analysis of Aqp8 and Adfp mrnas in colonic epithelial cells derived from wild-type and Cftr -/- mice treated with Ro (20 mg/kg/d for 5 days) or maintained on a control diet (n=10 mice per group). mrna levels are normalized to Gapdh and expressed relative to untreated wild-type cells. (b) Western blot of PPARγ using colonic epithelial cell lysates derived from Pparg f/f and Pparg IEC-/- mice. (c) Q-PCR analysis of mrnas shown in panel a in Pparg f/f and Pparg IEC-/- colonic epithelial cells (n=6 mice per group). Values are means ± s.e.m. For mice treated with Ro, P<0.01 and P<0.05 versus untreated mice of the same genotype. For untreated knock-out mice (Cftr -/- or Pparg IEC-/- ), P<0.01 and P<0.05 versus wild-type or Pparg f/f controls. Supplementary Figure 3. Genes up-regulated in Cftr- and Pparg-deficient mice. (a) Q- PCR analysis of Reg3b, Reg3g, Cxcl1, and Cxcl2 mrnas in colonic epithelial cells derived from wild-type and Cftr -/- mice treated with Ro (20 mg/kg/d for 5 days) or maintained on a control diet (n=10 mice per group). mrna levels are normalized to Gapdh and expressed relative to untreated wild-type cells. (b) Q-PCR analysis of mrnas shown in panel a in Pparg f/f and Pparg IEC-/- colonic epithelial cells (n=6 mice per group). Values are means ± s.e.m. For mice treated with Ro, P<0.01 and P<0.05 versus untreated mice of the same genotype. For untreated knock-out mice (Cftr -/- or Pparg IEC-/- ), P<0.01 and P<0.05 versus wild-type or Pparg f/f controls.

7 Supplementary Figure 4. Deletion of PPARγ exacerbates the phenotype of Cftr -/- mice. (a) Weight (grams) of male and female wild-type and Cftr -/- mice with combined Pparg f/f or Pparg IEC-/- genotypes demonstrated significantly reduced weight (age 30 days, 10 mice per group) by the combined deletion (Cftr -/- ;Pparg IEC-/- ) compared to control mice (Cftr -/- ;Pparg f/f ). There was no difference in the weight of Pparg IEC-/- compared to control Pparg f/f mice. Values are means ± s.e.m. P<0.001 and P<0.01. (b) Representative histology demonstrated alcian blue-staining mucin accumulation at the point of colonic obstruction in Cftr -/- ;Pparg IEC-/- mice. Supplementary Figure 5. Ro effect on PPARγ target genes in IB3-1 and S9 cells. (a) ANGPTL4 and ADFP mrnas are reduced in human CFTR mutant bronchial epithelial cell line IB3-1 and expression is restored by treatment with Ro. Values are means ± s.d. For cells treated with Ro, P<0.01 versus untreated cells and P<0.01 IB3-1 versus S9 control cells. Supplementary Figure 6. PPARγ agonist rosiglitazone (Ro) does not affect calciumdependent chloride secretion. (a) Ussing chamber experiments from wild-type or sibling Cftr -/- mice treated with or without Ro demonstrated reduced responses to carbachol (100 µm) in Cftr -/- colon and no difference in the response in Cftr -/- pre-treated for 5 days with Ro. (b) Pre-treatment with Ro for 48 hrs did not change the response to carbachol in T84 cells mounted in mucosal and serosal Ringer s solution. (c) Left panel; Pretreatment with CFTR inh -172 in amphotericin permeabilized T84 cells significantly reduced the peak current response to forskolin (10 µm) consistent with CFTR inhibition. Right panel; Ro did not affect the carbachol-responsive current in CFTR inhibited cells (p=0.12). Values are means ± s.e.m. P<0.01 and P< Results are representative of n=7 mice per group or n=6 T84 inserts per group.

8 Supplementary Figure 7. Quantitative expression for candidate ion transporter genes in Pparg f/f and Pparg IEC-/- mice treated with or without rosiglitazone (Ro). The results demonstrated increased expression of the basolaterally localized Slc4a2 (AE2, chloridebicarbonate exchanger 2, P<0.05) by rosiglitazone in Pparg f/f mice and increased expression for Slc26a6 (PAT1, chloride-bicarbonate exchanger) in Pparg IEC-/- mice (p<0.05). The expression of other candidate genes was unchanged. Slc4a1 (AE1) and Slc12a1 (NKCC2) were not present in colonic epithelial cells. mrna levels are normalized to Gapdh. Expression for Slc26a6 was 200x less than Slc26a3 (DRA1, chloride-bicarbonate exchanger) suggesting that DRA1 is the major apical chloridebicarbonate exchanger in the colon. Supplementary Figure 8. PPARγ binding sites function to enhance promoter activity. COS-7 cells were transiently transfected with control vector DNA (empty) or vector containing the previously identified PPARγ binding site for Angptl4 and the novel sites for Acaa1b, Mgll, and Car4 upstream of a minimal thymidine kinase TATA like promoter (ptal) directing firefly luciferase expression. Values are means ± s.d. of n=3 technical replicates (P<0.01). Results are representative of 3 independent biological replicates.

9 a Down-regulated genes GO Term p value Benjamini Biological Process Lipid metabolism Carboxylic acid metabolism 5.0e-8 2.3e-9 2.6e-4 3.0e-6 b 388 down-regulated genes in Cftr-/- 107 up by Ro Molecular Function Oxidoreductase activity 9.3e e-6 Up-regulated genes GO Term p value Benjamini Biological Process Inflammatory responses Response to wounding 1.5e-5 4.6e-5 8.6e-3 2.3e up-regulated genes in Cftr-/- 75 down by Ro Molecular Function Actin binding 2.6e-8 3.5e-5 Harmon, et al., Supplementary Fig. 1

10 a Aqp8 Relative expression Ro Cftr -/- Adfp Relative expression Ro Wildtype Wildtype Cftr -/- b Genotype Pparg f/f Pparg IEC-/- PPARγ βactin c Aqp8 Relative expression Ro NS Pparg f/f Pparg IEC-/- Adfp Relative expression Ro NS Pparg f/f Pparg IEC-/- Harmon, et al., Supplementary Fig. 2

11 a Reg3b Relative expression Ro Cftr -/- Wildtype Cftr -/- Reg3g Relative expression Ro Wildtype b Cxcl1 Relative expression Reg3b Relative expression Ro Ro Cftr -/- Wildtype Cxcl2 Relative expression Reg3g Relative expression Cftr -/- NS Pparg f/f Pparg IEC-/- Pparg f/f Pparg IEC/-/ Ro Ro Wildtype NS 5.5 NS Cxcl1 Relative expression Ro Pparg f/f NS Pparg IEC-/- Cxcl2 Relative expression Ro NS Pparg f/f NS Pparg IEC-/- Harmon, et al., Supplementary Fig. 3

12 a Grams Weight (male) Pparg f/f Pparg IEC-/- Cftr -/- ;Pparg f/f -/- Cftr ;Pparg IEC-/- Grams Weight (female) Pparg f/f Pparg IEC-/- Cftr -/- ;Pparg f/f -/- Cftr ;Pparg IEC-/- b CFTR DKO Harmon, et al., Supplementary Fig. 4

13 ANGPTL4 Relative expression Ro - S Ro - - IB3-1 ADFP Relative expression S9 IB3-1 Harmon, et al., Supplementary Fig. 5

14 a b Isc (µa cm -2 ) NS Isc (µa cm -2 ) NS 0 Ro Wildtype Cftr -/- 0 Ro c Isc (µa cm -2 ) Isc (µa cm -2 ) NS 0 DMSO CFTR inh Ro - - DMSO CFTR inh 172 Harmon, et al., Supplementary Fig. 6

15 Normalized gene expression Pparg f/f f/f Pparg Ro Pparg IEC-/- IEC-/- Pparg Ro Cftr Slc4a2 Slc4a4 Slc4a7 Slc9a1 Slc9a2 Slc9a3 Slc12a2 Slc26a3 Slc26a6 Scnnla Scnnlb Scnnlg Harmon, et al., Supplementary Fig. 7

16 PPRE PPARγ Enhancer Luciferase TATA Luciferase Normalized Fold Change Ro PPARγ Acaa1b -3.8k b Angptl4 intron 3 Mgll -7.3 kb Car4 intron 1 NSNSNS empty Harmon, et al., Supplemental Fig. 8

17 rank = rank based on false discovery analysis <FP> = false positive rate FDR = false discovery rate log2 (ko/wt) = log scale 2 (mean ko std chow / mean wt std chow) log 2 (koro/ko) = log scale 2 (mean ko ro chow / mean ko std chow) log 2 ttest ko/wt = unpaired t-test for wt std chow (log2 wt1, log2 wt2, log2 wt3, log2 wt4) versus ko std chow (log2 ko1, log2 ko2, log2 ko3, log2 ko4) log 2 ttest koro/ko = unpaired t-test for ko ro chow (log2 koro1, log2 koro2, log2 koro3, log2 koro4) versus ko std chow (log2 ko1, log2 ko2, log2 ko3, log2 ko4) Acc = GenBank Accession number Description = GenBank description rank <FP> FDR log2 (ko/wt) log2 (koro/ko) log2 ttest ko/wlog2 ttest koroacc Description E E NM cytochrome P450, family 4, subfamily b, polypeptide 1 (Cyp4b1) E E NM cystic fibrosis transmembrane conductance regulator homolog (Cftr) E E NM cytochrome P450, family 2, subfamily d, polypeptide 26 (Cyp2d26) E E NM cytochrome P450, family 4, subfamily f, polypeptide 14 (Cyp4f14) E E NM solute carrier family 16 (monocarboxylic acid transporters), member 9 (Slc16a9) E E NM cytochrome P450, family 2, subfamily d, polypeptide 26 (Cyp2d26) E E NM hydroxyacid oxidase (glycolate oxidase) 3 (Hao3) E E NM cytochrome P450, family 3, subfamily a, polypeptide 13 (Cyp3a13) E E NM cytochrome P450, family 3, subfamily a, polypeptide 13 (Cyp3a13) E E NM amnionless (Amn) E E NM prolactin receptor (Prlr) E E NM bone morphogenetic protein 3 (Bmp3) E E NM DNA segment, human D4S114 (D0H4S114) E E NM neurotensin (Nts) E E NM syncollin (Sycn) E E NM sulfotransferase family 1A, phenol-preferring, member 1 (Sult1a1) E E NM histocompatibility 2, class II antigen A, alpha (H2-Aa) E E NM C lectin-related protein A (Clra) E E NM tubulointerstitial nephritis antigen (Tinag) E E NM cytochrome P450, family 2, subfamily d, polypeptide 10 (Cyp2d10) E E NM afamin (Afm) E E NM cdna sequence BC (BC016235) E E NM RIKEN cdna G17 gene ( G17Rik) E E NM glucagon (Gcg) E E NM serum amyloid A 3 (Saa3) E E NM aldehyde dehydrogenase 1 family, member B1 (Aldh1b1) E E NM meprin 1 alpha (Mep1a) E E NM solute carrier family 3, member 1 (Slc3a1) E E NM histocompatibility 2, class II antigen A, beta 1 (H2-Ab1) E E NM angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2) E E NM dipeptidase 1 (renal) (Dpep1) E E NM expressed sequence AU (AU018778) E E NM UDP glycosyltransferase 1 family, polypeptide A6 (Ugt1a6) E E NM solute carrier family 22 (organic cation transporter), member 1 (Slc22a1)

18 E E NM response to metastatic cancers 1 (Rmcs1) E E NM RIKEN cdna D630035O19 gene (D630035O19Rik) E E NM neuraminidase 1 (Neu1) E E NM sialyltransferase 4C (beta-galactoside alpha-2,3-sialytransferase) (Siat4c) E E NM Ia-associated invariant chain (Ii) E E NM carboxylesterase 1 (Ces1) E E NM metallothionein 2 (Mt2) E E NM histocompatibility 2, class II antigen A, beta 1 (H2-Ab1) E E NM deiodinase, iodothyronine, type I (Dio1) E E NM cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22) E E NM alcohol dehydrogenase 5 (class III), chi polypeptide (Adh5) E E NM RIKEN cdna J23 gene ( J23Rik) E E NM neighbor of Punc E11 (Nope) E E NM transcription elongation factor A (SII), 3 (Tcea3) E E NM histocompatibility 2, class II antigen E beta (H2-Eb1) E E NM progressive ankylosis (Ank) E E NM cytochrome P450, family 3, subfamily a, polypeptide 13 (Cyp3a13) E E NM UDP glucuronosyltransferase 1 family, polypeptide A6B (Ugt1a6b) E E NM UDP-glucuronosyltransferase 8 (Ugt8) E E XM cytidine deaminase (Cda) E E NM selenoprotein P, plasma, 1 (Sepp1) E E NM vav 3 oncogene (Vav3) E E NM RIKEN cdna G15 gene ( G15Rik) E E NM solute carrier family 39 (metal ion transporter), member 5 (Slc39a5) E E NM aquaporin 1 (Aqp1) E E NM metallothionein 1 (Mt1) E E NM solute carrier family 5 (sodium-dependent vitamin transporter), member 6 (Slc5a6) E E NM expressed sequence AI (AI788959) E E NM cdna sequence BC (BC022224) E E NM NACHT, leucine rich repeat and PYD containing 6 (Nalp6) E E NM RIKEN cdna C15 gene ( C15Rik) E E NM hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (Hmgcs2) E E XM E E NM chromogranin B (Chgb) E E NM retinol binding protein 2, cellular (Rbp2) E E NM arylacetamide deacetylase (esterase) (Aadac) E E NM doublecortin-like kinase 3 (Dclk3) E E NM claudin 15 (Cldn15) E E NM solute carrier family 20, member 1 (Slc20a1) E E NM cytochrome P450, family 2, subfamily c, polypeptide 55 (Cyp2c55) E E NM dodecenoyl-coenzyme A delta isomerase (3,2 trans-enoyl-coenyme A isomerase) (Dci) E E NM ferritin heavy chain 1 (Fth1) E E NM endothelin 2 (Edn2) E E NM RIKEN cdna D17 gene ( D17Rik) E E NM RIKEN cdna A20 gene ( A20Rik) E E NM tubulointerstitial nephritis antigen (Tinag)

19 E E NM serum amyloid A 2 (Saa2) E E XM cubilin (intrinsic factor-cobalamin receptor) (Cubn) E E NM proline-rich acidic protein 1 (Prap1) E E NM cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (Cdkn2b) E E NM solute carrier family 8 (sodium/calcium exchanger), member 1 (Slc8a1) E E NM ectonucleoside triphosphate diphosphohydrolase 5 (Entpd5) E E NM serum amyloid A 1 (Saa1) E E NM carbonic anhydrase 4 (Car4) E E NM gamma-glutamyltransferase 1 (Ggt1) E E NM RIKEN cdna D02 gene ( D02Rik) E E NM glucosidase beta 2 (Gba2) E E NM zinc finger, FYVE domain containing 21 (Zfyve21) E E NM Tnf receptor-associated factor 1 (Traf1) E E NM hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A thiolase/ (Hadhb) E E NM small nuclear ribonucleoprotein N (Snrpn) E E NM RIKEN cdna C07 gene ( C07Rik) E E NM UDP glycosyltransferase 1 family, polypeptide A6 (Ugt1a6) E E NM toll-like receptor 2 (Tlr2) E E NM fumarylacetoacetate hydrolase domain containing 1 (Fahd1) E E NM vitamin D receptor (Vdr) E E NM RIKEN cdna E05 gene ( E05Rik) E E NM pancreatic polypeptide (Ppy) E E NM ring finger protein 128 (Rnf128) E E NM RIKEN cdna O03 gene ( O03Rik) E E NM glutamic pyruvic transaminase 1, soluble (Gpt1) E E NM lipoprotein lipase (Lpl) E E NM aldo-keto reductase family 7, member A5 (aflatoxin aldehyde reductase) (Akr7a5) E E NM cytochrome c oxidase, subunit VIIa 1 (Cox7a1) E E NM selenium binding protein 2 (Selenbp2) E E NM mercaptopyruvate sulfurtransferase (Mpst) E E NM aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1) E E NM scinderin (Scin) E E XM RIKEN cdna C14 gene ( C14Rik) E E NM RIKEN cdna O03 gene ( O03Rik) E E NM secretin (Sct) E E NM transforming growth factor beta 1 induced transcript 4 (Tgfb1i4) E E NM SNRPN upstream reading frame (Snurf) E E NM nucleosome binding protein 1 (Nsbp1) E E NM somatostatin (Sst) E E NM adipose differentiation related protein (Adfp) E E NM arginase type II (Arg2) E E NM quininoid dihydropteridine reductase (Qdpr) E E NM transmembrane 4 superfamily member 5 (Tm4sf5) E E NM sulfotransferase family 1D, member 1 (Sult1d1) E E NM transmembrane protein 25 (Tmem25) E E NM cyclin-dependent kinase inhibitor 1C (P57) (Cdkn1c)

20 E E NM suppressor of cytokine signaling 2 (Socs2) E E NM transforming growth factor beta 1 induced transcript 4 (Tgfb1i4) E E NM RIKEN cdna H05 gene ( H05Rik) E E NM cytochrome P450, family 4, subfamily v, polypeptide 3 (Cyp4v3) E E NM G protein-coupled receptor 49 (Gpr49) E E NM purinergic receptor P2Y, G-protein coupled 1 (P2ry1) E E NM hydroxyacyl-coenzyme A dehydrogenase/3-ketoacyl-coenzyme A thiolase/enoyl-t (Hadhb) E E NM phospholipid scramblase 4 (Plscr4) E E NM FERM, RhoGEF and pleckstrin domain protein 2 (Farp2) E NM kallikrein 27 (Klk27) E NM dipeptidylpeptidase 4 (Dpp4) E NM inner mitochondrial membrane peptidase 2-like (S. cerevisiae) (Immp2l) E NM hydroxyacyl glutathione hydrolase (Hagh) E NM prion protein (Prnp) E NM selenium binding protein 1 (Selenbp1) E NM trehalase (brush-border membrane glycoprotein) (Treh) E NM peroxisomal biogenesis factor 11a (Pex11a) E NM chloride channel calcium activated 3 (Clca3) E NM solute carrier family 5 (sodium/glucose cotransporter), member 1 (Slc5a1) E NM UDP glucuronosyltransferase 1 family, polypeptide A6B (Ugt1a6b) E NM small nuclear ribonucleoprotein N (Snrpn) E NM peroxisomal membrane protein 4 (Pxmp4) E NM voltage-dependent anion channel 3 (Vdac3) E NM galactosidase, beta 1 (Glb1) E NM enoyl coenzyme A hydratase 1, peroxisomal (Ech1) E NM UDP glucuronosyltransferase 1 family, polypeptide A7C (Ugt1a7c) E NM SWAP complex protein (Swap70) E NM RIKEN cdna B20 gene ( B20Rik) E NM olfactory receptor 165 (Olfr165) E NM solute carrier family 22 (organic cation transporter), member 1 (Slc22a1) E NM c-mer proto-oncogene tyrosine kinase (Mertk) E NM RIKEN cdna L02 gene ( L02Rik) E NM L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain (Hadhsc) E XM E NM gene rich cluster, C10 gene (Grcc10) E NM ankyrin 3, epithelial (Ank3), transcript variant E NM expressed sequence AI (AI987692) E NM nuclear receptor subfamily 1, group H, member 3 (Nr1h3) E NM RIKEN cdna C23 gene ( C23Rik) E NM deiodinase, iodothyronine, type I (Dio1) E NM calmin (Clmn) E NM mucolipin 1 (Mcoln1) E XM cytochrome P450, family 2, subfamily c, polypeptide 66 (Cyp2c66) E NM UDP glycosyltransferase 1 family, polypeptide A10 (Ugt1a10) E NM DNA segment, Chr 10, ERATO Doi 214, expressed (D10Ertd214e) E NM UDP glycosyltransferase 1 family, polypeptide A6 (Ugt1a6)

21 E NM transglutaminase 3, E polypeptide (Tgm3) E NM small EDRK-rich factor 1 (Serf1) E NM abhydrolase domain containing 1 (Abhd1) E NM Rho-related BTB domain containing 3 (Rhobtb3) E XM RIKEN cdna F06 gene ( F06Rik) E XM RIKEN cdna O07 gene ( O07Rik) E NM annexin A13 (Anxa13) E NM kallikrein 5 (Klk5) E NM G protein-coupled receptor 172B (Gpr172b) E NM ceroid lipofuscinosis, neuronal 3, juvenile (Batten, Spielmeyer-Vogt disease) (Cln3) E NM kallikrein 27 (Klk27) E NM vitamin D receptor (Vdr) E NM low density lipoprotein receptor-related protein 4 (Lrp4) E NM ring finger protein 152 (Rnf152), transcript variant E NM beta-2 microglobulin (B2m) E NM RIKEN cdna P03 gene ( P03Rik) E NM RIKEN cdna H14 gene ( H14Rik) E NM cdna sequence BC (BC038286) E NM transient receptor potential cation channel, subfamily M, member 5 (Trpm5) E NM colipase, pancreatic (Clps) E NM RUN domain containing 3B (Rundc3b) E NM RIKEN cdna O21 gene ( O21Rik) E NM RIKEN cdna B930062P21 gene (B930062P21Rik) E NM cathepsin S (Ctss) E NM hypothetical protein C730031G17 (C730031G17) E NM alanyl (membrane) aminopeptidase (Anpep) E NM heat shock protein, A (Hspa9a) E NM melanoma inhibitory activity 1 (Mia1) E NM transmembrane protein 30B (Tmem30b) E NM regulated endocrine-specific protein 18 (Resp18) E NM pyrroline-5-carboxylate reductase-like (Pycrl) E XM RIKEN cdna G18 gene ( G18Rik) E NM RIKEN cdna B05 gene ( B05Rik) E NM hydroxysteroid (17-beta) dehydrogenase 2 (Hsd17b2) E NM RIKEN cdna E130201N16 gene (E130201N16Rik) E NM mitochondrial carrier homolog 2 (C. elegans) (Mtch2) E NM RIKEN cdna L17 gene ( L17Rik) E NM erythrocyte protein band 4.1-like 3 (Epb4.1l3) E NM pleckstrin homology domain-containing, family A member 3 (Plekha3) E NM RIKEN cdna G23 gene ( G23Rik) E NM meprin 1 beta (Mep1b) E NM nerve growth factor, alpha (Ngfa) E NM DNA segment, Chr 11, Brigham & Womens Genetics 0434 expressed (D11Bwg0434e) E NM RIKEN cdna A130092J06 gene (A130092J06Rik) E NM expressed sequence AI (AI415282) E NM transient receptor potential cation channel, subfamily M, member 6 (Trpm6)

22 E NM tweety homolog 3 (Drosophila) (Ttyh3) E XM E NM crystallin, lamda 1 (Cryl1) E NM phosphoglucomutase 2 (Pgm2) E XM RIKEN cdna J22 gene ( J22Rik) E NM glycerophosphodiester phosphodiesterase domain containing 2 (Gdpd2) E NM neuropathy target esterase (Nte) E NM adenylate cyclase 6 (Adcy6) E NM acetyl-coenzyme A dehydrogenase, medium chain (Acadm) E NM abhydrolase domain containing 1 (Abhd1) E NM basic transcription element binding protein 1 (Bteb1) E NM acetyl-coenzyme A acyltransferase 1A (Acaa1a) E XM RIKEN cdna F06 gene ( F06Rik) E NM sirtuin 3 (silent mating type information regulation 2, homolog) 3 (S. cerevisiae) (Sirt3) E NM RIKEN cdna O03 gene ( O03Rik) E NM carbonic anhydrase 2 (Car2) E NM lactation elevated 1 (Lace1) E NM RIKEN cdna P08 gene ( P08Rik) E NM expressed sequence AI (AI415282) E XM RIKEN cdna P09 gene ( P09Rik) E XM RIKEN cdna I15 gene ( I15Rik) E NM RIKEN cdna B02 gene ( B02Rik) E XM eukaryotic translation initiation factor 4A2 (Eif4a2) E NM solute carrier family 22 (organic cation transporter), member 5 (Slc22a5) E NM osteoclast inhibitory lectin (Ocil) E NM acetyl-coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase) (Acaa2) E NM diacylglycerol O-acyltransferase 1 (Dgat1) E NM basigin (Bsg) E NM RIKEN cdna I22 gene ( I22Rik) E NM aquaporin 11 (Aqp11) E NM vav 3 oncogene (Vav3) E NM RIKEN cdna E130307M08 gene (E130307M08Rik) E NM copper chaperone for superoxide dismutase (Ccs) E NM CD83 antigen (Cd83) E XM activin A receptor, type IC (Acvr1c) E NM toll-like receptor 5 (Tlr5) E NM EST AA (AA881470) E NM guanine nucleotide binding protein, alpha 11 (Gna11) E NM cytochrome P450, family 2, subfamily d, polypeptide 34 (Cyp2d34) E NM kallikrein 8 (Klk8) E NM homeo box B4 (Hoxb4) E NM RIKEN cdna K24 gene ( K24Rik) E NM ubiquitin D (Ubd) E NM RIKEN cdna I15 gene ( I15Rik) E NM hypothetical protein F830003B07 (F830003B07) E NM cytochrome P450, family 4, subfamily f, polypeptide 13 (Cyp4f13)

23 E NM zinc finger protein 292 (Zfp292) E NM RIKEN cdna D930010J01 gene (D930010J01Rik) E NM nidogen 2 (Nid2) E NM cytochrome c oxidase, subunit VIIa 2 (Cox7a2) E XM E NM small EDRK-rich factor 2 (Serf2) E XM E NM RIKEN cdna C01 gene ( C01Rik) E NM RIKEN cdna N20 gene ( N20Rik) E XM E NM RIKEN cdna N20 gene ( N20Rik) E NM SPRY domain-containing SOCS box 4 (Ssb4) E NM tripartite motif protein 2 (Trim2) E NM isocitrate dehydrogenase 3 (NAD) beta (Idh3b) E NM protein tyrosine phosphatase-like (proline instead of catalytic arginine), member a (Ptpla) E NM RIKEN cdna O13 gene ( O13Rik) E NM ectonucleoside triphosphate diphosphohydrolase 2 (Entpd2) E NM expressed in non-metastatic cells 3 (Nme3) E XM E NM O-linked N-acetylglucosamine (GlcNAc) transferase(ogt) E NM solute carrier organic anion transporter family, member 2b1 (Slco2b1) E NM histocompatibility 2, blastocyst (H2-Bl) E NM glutamate-cysteine ligase, modifier subunit (Gclm) E NM splicing factor, arginine/serine-rich 7 (Sfrs7) E NM nuclear factor I/C (Nfic) E NM adenosine kinase (Adk) E NM ADP-ribosylation factor-like 3 (Arl3) E NM RIKEN cdna N04 gene ( N04Rik) E NM zinc finger protein 637 (Zfp637) E NM hydroxyprostaglandin dehydrogenase 15 (NAD) (Hpgd) E NM RIKEN cdna N19 gene ( N19Rik) E NM peroxisome biogenesis factor 19 (Pex19) E NM deiodinase, iodothyronine, type I (Dio1) E NM RIKEN cdna J14 gene ( J14Rik) E NM uroporphyrinogen decarboxylase (Urod) E NM flavin containing monooxygenase 5 (Fmo5) E NM heterogeneous nuclear ribonucleoprotein methyltransferase-like 1 (S. cerevisiae) (Hrmt1l1) E NM RIKEN cdna A14 gene ( A14Rik) E XM sorting nexin 7 (Snx7) E NM serine protease inhibitor, Kunitz type 2 (Spint2) E NM amyotrophic lateral sclerosis 2 (juvenile) chromosome region, candidate 2 homolog (Als2cr2) E NM interferon gamma inducible protein 30 (Ifi30) E NM thiosulfate sulfurtransferase, mitochondrial (Tst) E XM cytochrome P450, family 2, subfamily c, polypeptide 65 (Cyp2c65) E NM histocompatibility 2, class II, locus Mb1 (H2-DMb1) E NM solute carrier family 4 (anion exchanger), member 2 (Slc4a2)

24 E NM RIKEN cdna H13 gene ( H13Rik) E NM RIKEN cdna B430218L07 gene (B430218L07Rik) E NM ORM1-like 3 (S. cerevisiae) (Ormdl3) E NM hydroxysteroid 11-beta dehydrogenase 2 (Hsd11b2) E NM WD repeat domain 9 (Wdr9) E NM cytochrome P450, family 4, subfamily f, polypeptide 16 (Cyp4f16) E NM RIKEN cdna C14 gene ( C14Rik) E NM Down syndrome critical region homolog 5 (human) (Dscr5) E NM RIKEN cdna B02 gene ( B02Rik) E NM surfeit gene 1 (Surf1) E NM RIKEN cdna N19 gene ( N19Rik) E NM carboxypeptidase D (Cpd) E NM RIKEN cdna G13 gene ( G13Rik) E NM grancalcin (Gca) E NM CUE domain containing 2 (Cuedc2) E XM sestrin 1 (Sesn1) E XM RIKEN cdna A05 gene ( A05Rik) E NM single-stranded DNA binding protein 2 (Ssbp2) E NM creatine kinase, mitochondrial 2 (Ckmt2) E NM DiGeorge syndrome critical region gene 6 (Dgcr6) E NM utrophin (Utrn) E XM E NM chromogranin A (Chga) E NM succinate dehydrogenase complex, subunit B, iron sulfur (Ip) (Sdhb) E NM amiloride binding protein 1 (amine oxidase, copper-containing) (Abp1) E NM sirtuin 3 (silent mating type information regulation 2, homolog) 3 (S. cerevisiae) (Sirt3) E NM cdna sequence BC (BC003479) E NM RIKEN cdna N17 gene ( N17Rik) E NM RIKEN cdna G13 gene ( G13Rik) E NM zinc finger protein 292 (Zfp292) E NM propionyl Coenzyme A carboxylase, beta polypeptide (Pccb) E NM RIKEN cdna D430015B01 gene (D430015B01Rik) E NM ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) E NM hexosaminidase B (Hexb) E NM cysteine and histidine rich 1 (Cyhr1) E NM RIKEN cdna H11 gene ( H11Rik) E NM SRY-box containing gene 4 (Sox4) E NM ATPase, Na/K transporting, beta 1 polypeptide (Atp1b1) E NM microtubule associated serine/threonine kinase 2 (Mast2) E NM adducin 3 (gamma) (Add3) E NM propionyl-coenzyme A carboxylase, alpha polypeptide (Pcca) E NM trans-acting transcription factor 4 (Sp4) E NM isocitrate dehydrogenase 3 (NAD), gamma (Idh3g) E NM RIKEN cdna L19 gene ( L19Rik) E NM RIKEN cdna N07 gene ( N07Rik) E NM solute carrier family 23 (nucleobase transporters), member 2 (Slc23a2)