Supplemental Figures and Tables

Transcript

1 KLF2 and KLF4 control endothelial identity and vascular integrity Panjamaporn Sangwung 1, 2,#, Guangjin Zhou 1,#, Lalitha Nayak 1,3, E. Ricky Chan 4, Sandeep Kumar 5, Dong-Won Kang 5, Rongli Zhang 1, Xudong Liao 1, Yuan Lu 1, Keiki Sugi 1, Hisashi Fujioka 7, Hong Shi 1, Stephanie D Lapping 1, Chandra C. Ghosh 8, Sarah J. Higgins 8, Samir M. Parikh 8, Hanjoon Jo 5,6, and Mukesh K Jain 1,2,9 Supplemental Figures and Tables 1

2 Supplemental Figure 1 Day 6 post-tamoxifen Day 5 post-tamoxifen Day 4 post-tamoxifen A Relative mrna expression B Relative mrna expression C Relative mrna expression D CRE Klf2 ** NS ** CRE EC-Klf2-KO EC-Klf4-KO EC-DKO KLF4 Klf4 NS ** ** Nos3 Thbd ** NS NS Klf2 Klf4 Nos3 Thbd ** 3 ** 3 ** 3 ** NS ** ** ** ** NS NS * CRE EC-Klf2-KO EC-Klf4-KO Klf2 Klf4 Nos3 Thbd ** 3 ** ** ** NS ** 3 3 NS NS ** NS NS NS CRE EC-Klf2-KO EC-Klf4-KO EC-DKO EC-DKO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO ** * * CRE EC-Klf2-KO EC-Klf4-KO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO EC-DKO CRE EC-Klf2-KO EC-Klf4-KO EC-DKO TM GAPDH enos GAPDH Supplemental Figure 1. Expression of Klf2, Klf4, and their targets in primary cardiac microvascular EC. Relative mrna expression levels of Klf2 (Kruppel-like family of transcription factor 2), Klf4, Nos3 (endothelial nitric oxide synthase), and Thbd (thrombomodulin) in primary cardiac microvascular EC isolated from mice at day 6 (A), day 5 (B), and day 4 (C) following tamoxifen (n=3-5 per genotype, each sample was pooled from 2 mice). (D) Representative western blot of primary cardiac microvascular EC using antibodies against KLF4, enos (endothelial nitric oxide synthase), TM (thrombomodulin), and GAPDH (endogenous glyceraldehyde 3-phosphate dehydrogenase). CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout, EC-Klf2-KO: EC-specific Klf2 knockout, EC-Klf4-KO: EC-specific Klf4 knockout. Sample in each group was pooled from 7-8 mice. *: P < 0.05, * *: P < 0.01, NS: Not significant. One-way ANOVA with Bonferroni's post hoc test. 2

3 Supplemental Figure Baseline Day 6 post-tamoxifen EKG (mv) CRE EKG (mv) EC-DKO ST EKG (mv) EC-Klf2-KO EKG (mv) EC-Klf4-KO ms Supplemental Figure 2. Electrocardiogram (EKG) recording. Representative EKG of CRE, EC-DKO, EC-Klf2-KO, and EC-Klf4-KO mice at baseline without tamoxifen (left) and day 6 post-tamoxifen injection (right). n=4-5 per genotype. CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout, EC-Klf2-KO: EC-specific Klf2 knockout, EC-Klf4-KO: EC-specific Klf4 knockout. 3

4 Supplemental Figure 3 A Cardiac function 60 NS ** NS 80 NS ** NS 40 NS NS p= ** ** FS (%) EF (%) CO (ml/min) CRE EC-Klf2-KO EC-Klf4-KO EC-DKO 0 CRE EC-Klf2-KO EC-Klf4-KO EC-DKO 0 CRE EC-Klf2-KO EC-Klf4-KO EC-DKO B 5 NS ** NS C 5 NS NS * Chamber size 5 NS ** NS LV/BW (mg/g) LVIDd (mm) LVIDs (mm) CRE EC-Klf2-KO EC-Klf4-KO EC-DKO 0 CRE EC-Klf2-KO EC-Klf4-KO EC-DKO 0 CRE EC-Klf2-KO EC-Klf4-KO EC-DKO Supplemental Figure 3. Echocardiographic analysis at day 6 after tamoxifen administration. (A) Fractional shortening (FS), ejection fraction (EF), and cardiac output (CO) in CRE (n=3), EC-Klf2-KO (4), EC-Klf4-KO (n=4), and EC-DKO mice (n=5). (B) Left ventricle (LV)-to-body weight (BW) ratio. (C) LV internal dimension at end diastole (LVIDd), and at end systole (LVIDs). CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout, EC-Klf2-KO: EC-specific Klf2 knockout, EC-Klf4-KO: EC-specific Klf4 knockout. Data are presented as mean ± SEM. *: P < 0.05, * *: P < NS: Not significant. One-way ANOVA with Bonferroni's post hoc test. 4

5 Supplemental Figure 4 EC-Klf2-KO EC-Klf4-KO Brain Lung Heart Skin Supplemental Figure 4. Gross post-mortem examination in EC-Klf2-KO and EC-Klf4-KO mice. Representative gross anatomy pictures of brain, lungs, heart, and subcutaneous tissues from EC-Klf2-KO (EC-specific Klf2 knockout) and EC-Klf4-KO (EC-specific Klf4 knockout) mice at day 6 post-tamoxifen (n=3-4 per genotype). 5

6 Supplemental Figure 5 A Eosin (Lung) EM (Lung) Evans blue (Lung) Evans blue (Kidney) CRE EC-DKO B Permeability (corrected E620) C Permeability (corrected E620) D Permeability (corrected E620) Day 5 post-tamoxifen Lung 0.5 Kidney 0.5 Heart ** 0.4 * Day 4 post-tamoxifen Lung Kidney Heart * 0.5 * 0.5 NS CRE EC-DKO CRE EC-DKO CRE EC-DKO CRE EC-DKO CRE EC-DKO CRE EC-DKO Day 6 post-tamoxifen Lung * Kidney * Heart CRE EC-DKO CRE EC-DKO CRE EC-DKO NS * Supplemental Figure 5. Endothelial-specific deletion of Klf2 and Klf4 results in vascular leak. (A, top) Representative pictures of lung Eosin staining show erythrocyte leakage (white arrow) in extravascular tissues at day 6 post-tamoxifen. (n=3-4 per genotype). Scale bar is 200 μm. (A, middle) Representative electron microscopic (EM) images of lung indicate discontinuity of endothelial monolayer at day 6 post-tamoxifen (n=3-4 per genotype). Scale bar is 2 μm. Endothelial cells (EC): red arrowhead, extravascular erythrocytes: white arrow, leucocytes: blue arrow. (A, bottom) Representative pictures of Evans blue vascular permeability assay for the lungs and kidneys at day 6 post-tamoxifen (n=7-8 per genotype). (B-D) Quantification of extravasated Evans Blue dye in the lungs, kidneys, and heart at day 4 (B, n=6-8 per genotype), day 5 (C, n=6-9 per genotype), and day 6 (D, n=7-8 per genotype) post-tamoxifen. CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout. Data are presented as mean ± SEM. *: P < 0.05, * *: P < 0.01, NS: Not significant. 2-tailed Student s t test. 6

7 Supplemental Figure 6 5 NS * ** CRE EC-DKO Relative mrna expression F3 Serpine1 F2rl3 Supplemental Figure 6. Expression of F3, Serpine1, and F2rl3 mrna in primary cardiac microvascular EC at day 6 post-tamoxifen. n=3-4 per genotype, each sample was pooled from 2 mice. F3: tissue factor, Serpine1: serine (or cysteine) peptidase inhibitor, clade E, member 1, F2rl3: coagulation factor II receptor-like 3, CRE: Cdh5(PAC)-Ert2cre, EC-DKO: EC-specific Klf2 and Klf4 double knockout. *: P < 0.05, * *: P < 0.01, NS: Not significant. 2-tailed Student s t test. 7

8 Supplemental Figure 7 EC-DKO EC-DKO EC-DKO EM (Brain) Supplemental Figure 7. Representative electron microscopic (EM) images of brain indicate degeneration of EC and extravascular erythrocytes in the EC-DKO mice at day 6 post-tamoxifen. Scale bar is 2 μm. Endothelial cells (EC): red arrowhead, extravascular erythrocytes: white arrow, leucocytes: blue arrow, vessel lumen: black arrowhead (n=3-4 per genotype). EC-DKO: EC-specific Klf2 and Klf4 double knockout. 8

9 Supplemental Table 1. Differentially expressed genes (q<0.05) in cardiac microvascular EC obtained from EC-DKO in comparison to CRE mice at day 6 post-tamoxifen. Ensembl_ID Gene Description EC-DKOvsCRE.log2FC EC-DKOvsCRE.RawP EC-DKOvsCRE.FDR ENSMUSG Gm7809 predicted gene E E-177 ENSMUSG Mest mesoderm specific transcript E E-109 ENSMUSG Ces2e carboxylesterase 2E E E-87 ENSMUSG Syt7 synaptotagmin VII E E-83 ENSMUSG Alpl alkaline phosphatase, liver/bone/kidney E E-77 ENSMUSG Apln apelin E E-70 ENSMUSG Casp3 caspase E E-65 ENSMUSG Tnc tenascin C E E-59 ENSMUSG Dpysl3 dihydropyrimidinase-like E E-58 ENSMUSG Ptprr protein tyrosine phosphatase, receptor type, R E E-57 ENSMUSG H2-T24 histocompatibility 2, T region locus E E-57 ENSMUSG Cd109 CD109 antigen E E-57 ENSMUSG Gm25360 predicted gene, E E-51 ENSMUSG Ceacam1 carcinoembryonic antigen-related cell adhesion molecule E E-51 ENSMUSG Prg4 proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein) E E-50 ENSMUSG Sele selectin, endothelial cell E E-49 ENSMUSG Plau plasminogen activator, urokinase E E-46 ENSMUSG Tubb6 tubulin, beta 6 class V E E-45 ENSMUSG Ptchd1 patched domain containing E E-44 ENSMUSG Wisp1 WNT1 inducible signaling pathway protein E E-44 ENSMUSG Angpt2 angiopoietin E E-42 ENSMUSG Jam2 junction adhesion molecule E E-42 ENSMUSG Igf1 insulin-like growth factor E E-40 ENSMUSG Pcsk5 proprotein convertase subtilisin/kexin type E E-40 ENSMUSG Sparc secreted acidic cysteine rich glycoprotein E E-40 ENSMUSG Ahr aryl-hydrocarbon receptor E E-39 ENSMUSG Abcb4 ATP-binding cassette, sub-family B (MDR/TAP), member E E-39 ENSMUSG Slc30a4 solute carrier family 30 (zinc transporter), member E E-39 ENSMUSG Akr1c19 aldo-keto reductase family 1, member C E E-39 ENSMUSG Kcnj2 potassium inwardly-rectifying channel, subfamily J, member E E-39 ENSMUSG Nr3c2 nuclear receptor subfamily 3, group C, member E E-38 ENSMUSG Crispld1 cysteine-rich secretory protein LCCL domain containing E E-38 ENSMUSG Akr1b8 aldo-keto reductase family 1, member B E E-38 ENSMUSG Slc26a10 solute carrier family 26, member E E-37 ENSMUSG Prcp prolylcarboxypeptidase (angiotensinase C) E E-37 ENSMUSG Nuak1 NUAK family, SNF1-like kinase, E E-37 ENSMUSG Fam167b family with sequence similarity 167, member B E E-37 ENSMUSG Clca5 chloride channel calcium activated E E-36 ENSMUSG P05Rik RIKEN cdna P05 gene E E-36 ENSMUSG Vim vimentin E E-36 ENSMUSG Rassf9 Ras association (RalGDS/AF-6) domain family (N-terminal) member E E-36 ENSMUSG Tmsb10 thymosin, beta E E-35 ENSMUSG Tln2 talin E E-35 ENSMUSG Clmn calmin E E-35 ENSMUSG Tuba1c tubulin, alpha 1C E E-34 ENSMUSG Kank4 KN motif and ankyrin repeat domains E E-34 ENSMUSG Tll1 tolloid-like E E-33 ENSMUSG Myh10 myosin, heavy polypeptide 10, non-muscle E E-33 ENSMUSG Ces2g carboxylesterase 2G E E-33 ENSMUSG Rad51ap1 RAD51 associated protein E E-32 ENSMUSG Slc22a4 solute carrier family 22 (organic cation transporter), member E E-31 ENSMUSG Nrip2 nuclear receptor interacting protein E E-31 ENSMUSG Coro6 coronin E E-31 ENSMUSG Syt15 synaptotagmin XV E E-31 ENSMUSG Cxxc4 CXXC finger E E-31 ENSMUSG Aif1l allograft inflammatory factor 1-like E E-31 ENSMUSG Nepn nephrocan E E-30 ENSMUSG Map1b microtubule-associated protein 1B E E-30 ENSMUSG Nebl nebulette E E-29 ENSMUSG Pltp phospholipid transfer protein E E-29 ENSMUSG Cytl1 cytokine-like E E-29 ENSMUSG Acss1 acyl-coa synthetase short-chain family member E E-29 ENSMUSG Ankrd33b ankyrin repeat domain 33B E E-29 ENSMUSG Sqle squalene epoxidase E E-29 ENSMUSG Wnt9b wingless-type MMTV integration site family, member 9B E E-28 ENSMUSG Gm12216 predicted gene E E-28 ENSMUSG Slco1a4 solute carrier organic anion transporter family, member 1a E E-27 ENSMUSG Adamts17 a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, E E-27 ENSMUSG Dnm3 dynamin E E-27 ENSMUSG Cd276 CD276 antigen E E-27 ENSMUSG Magix MAGI family member, X-linked E E-27 Page 1 of 84

10 ENSMUSG Rasgrf2 RAS protein-specific guanine nucleotide-releasing factor E E-27 ENSMUSG Pitpnm3 PITPNM family member E E-27 ENSMUSG AU expressed sequence AU E E-27 ENSMUSG Gbp4 guanylate binding protein E E-27 ENSMUSG Pxdn peroxidasin homolog (Drosophila) E E-27 ENSMUSG Mamdc2 MAM domain containing E E-27 ENSMUSG Stmn2 stathmin-like E E-27 ENSMUSG Jazf1 JAZF zinc finger E E-26 ENSMUSG Slc28a2 solute carrier family 28 (sodium-coupled nucleoside transporter), member E E-26 ENSMUSG Thbd thrombomodulin E E-26 ENSMUSG Wbp5 WW domain binding protein E E-26 ENSMUSG Prrt4 proline-rich transmembrane protein E E-26 ENSMUSG Sidt1 SID1 transmembrane family, member E E-26 ENSMUSG Sybu syntabulin (syntaxin-interacting) E E-26 ENSMUSG Fbln5 fibulin E E-26 ENSMUSG E2f1 E2F transcription factor E E-26 ENSMUSG Rhpn2 rhophilin, Rho GTPase binding protein E E-26 ENSMUSG Cdkn1c cyclin-dependent kinase inhibitor 1C (P57) E E-26 ENSMUSG Lpar4 lysophosphatidic acid receptor E E-26 ENSMUSG S100a11 S100 calcium binding protein A11 (calgizzarin) E E-26 ENSMUSG Litaf LPS-induced TN factor E E-26 ENSMUSG Sgpp2 sphingosine-1-phosphate phosphotase E E-25 ENSMUSG Tmtc1 transmembrane and tetratricopeptide repeat containing E E-25 ENSMUSG Scn4b sodium channel, type IV, beta E E-25 ENSMUSG Dll1 delta-like 1 (Drosophila) E E-25 ENSMUSG Myof myoferlin E E-25 ENSMUSG Entpd1 ectonucleoside triphosphate diphosphohydrolase E E-25 ENSMUSG Txlnb taxilin beta E E-25 ENSMUSG Fstl1 follistatin-like E E-25 ENSMUSG Fndc5 fibronectin type III domain containing E E-25 ENSMUSG Mn1 meningioma E E-24 ENSMUSG Prom1 prominin E E-24 ENSMUSG Rell1 RELT-like E E-24 ENSMUSG Anxa1 annexin A E E-24 ENSMUSG Nid1 nidogen E E-24 ENSMUSG Arl4d ADP-ribosylation factor-like 4D E E-24 ENSMUSG Sdk1 sidekick homolog 1 (chicken) E E-24 ENSMUSG Acacb acetyl-coenzyme A carboxylase beta E E-24 ENSMUSG Gm14290 predicted gene E E-24 ENSMUSG Podxl podocalyxin-like E E-23 ENSMUSG Etv4 ets variant E E-23 ENSMUSG Sh2d5 SH2 domain containing E E-23 ENSMUSG Acan aggrecan E E-23 ENSMUSG Ywhah tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide E E-23 ENSMUSG Disp2 dispatched homolog 2 (Drosophila) E E-23 ENSMUSG Zfp185 zinc finger protein E E-23 ENSMUSG Cdc25c cell division cycle 25C E E-23 ENSMUSG Car13 carbonic anhydrase E E-23 ENSMUSG Mtmr11 myotubularin related protein E E-22 ENSMUSG Ccnb2 cyclin B E E-22 ENSMUSG Man1a mannosidase 1, alpha E E-22 ENSMUSG Mettl7a1 methyltransferase like 7A E E-22 ENSMUSG B4galt3 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide E E-22 ENSMUSG Slc6a6 solute carrier family 6 (neurotransmitter transporter, taurine), member E E-22 ENSMUSG Pgk1 phosphoglycerate kinase E E-22 ENSMUSG Kit kit oncogene E E-22 ENSMUSG Spice1 spindle and centriole associated protein E E-21 ENSMUSG Bmp2 bone morphogenetic protein E E-21 ENSMUSG Mbnl3 muscleblind-like 3 (Drosophila) E E-21 ENSMUSG Kifc2 kinesin family member C E E-21 ENSMUSG Npnt nephronectin E E-21 ENSMUSG Tmem56 transmembrane protein E E-21 ENSMUSG Atp1a2 ATPase, Na+/K+ transporting, alpha 2 polypeptide E E-21 ENSMUSG Lamb1 laminin B E E-21 ENSMUSG Dhrs3 dehydrogenase/reductase (SDR family) member E E-21 ENSMUSG Myoz2 myozenin E E-21 ENSMUSG Fkbp1a FK506 binding protein 1a E E-21 ENSMUSG Kcnk3 potassium channel, subfamily K, member E E-21 ENSMUSG Col13a1 collagen, type XIII, alpha E E-21 ENSMUSG Sh3d21 SH3 domain containing E E-21 ENSMUSG Nxpe2 neurexophilin and PC-esterase domain family, member E E-21 ENSMUSG St6galnac2 ST6 (alpha-n-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-n-acetylgalactosaminide alpha-2,6-sialyltransferase E E-21 ENSMUSG Hdac11 histone deacetylase E E-21 Page 2 of 84

11 ENSMUSG Sh3pxd2b SH3 and PX domains 2B E E-21 ENSMUSG Gm16070 predicted gene E E-21 ENSMUSG Mrgprh MAS-related GPR, member H E E-21 ENSMUSG Morf4l2 mortality factor 4 like E E-20 ENSMUSG Adamtsl1 ADAMTS-like E E-20 ENSMUSG Pbld1 phenazine biosynthesis-like protein domain containing E E-20 ENSMUSG Rassf2 Ras association (RalGDS/AF-6) domain family member E E-20 ENSMUSG Sacs sacsin E E-20 ENSMUSG Cd93 CD93 antigen E E-20 ENSMUSG Slc38a11 solute carrier family 38, member E E-20 ENSMUSG Gbp5 guanylate binding protein E E-20 ENSMUSG Nus1 nuclear undecaprenyl pyrophosphate synthase 1 homolog (S. cerevisiae) E E-20 ENSMUSG Arhgap11a Rho GTPase activating protein 11A E E-20 ENSMUSG Arf2 ADP-ribosylation factor E E-20 ENSMUSG Pde4b phosphodiesterase 4B, camp specific E E-20 ENSMUSG Vegfc vascular endothelial growth factor C E E-20 ENSMUSG Slc12a7 solute carrier family 12, member E E-19 ENSMUSG Plp2 proteolipid protein E E-19 ENSMUSG Nts neurotensin E E-19 ENSMUSG Kazald1 Kazal-type serine peptidase inhibitor domain E E-19 ENSMUSG Sgms2 sphingomyelin synthase E E-19 ENSMUSG Mfsd7c major facilitator superfamily domain containing 7C E E-19 ENSMUSG Ulbp1 UL16 binding protein E E-19 ENSMUSG Pvr poliovirus receptor E E-19 ENSMUSG Cyp27a1 cytochrome P450, family 27, subfamily a, polypeptide E E-19 ENSMUSG Prnp prion protein E E-19 ENSMUSG Pygm muscle glycogen phosphorylase E E-18 ENSMUSG Rtn4 reticulon E E-18 ENSMUSG Ppa1 pyrophosphatase (inorganic) E E-18 ENSMUSG Plk1 polo-like kinase E E-18 ENSMUSG Itga2 integrin alpha E E-18 ENSMUSG Arhgef26 Rho guanine nucleotide exchange factor (GEF) E E-18 ENSMUSG Chst1 carbohydrate (keratan sulfate Gal-6) sulfotransferase E E-18 ENSMUSG Zdhhc15 zinc finger, DHHC domain containing E E-18 ENSMUSG J21Rik1 RIKEN cdna J21 gene E E-18 ENSMUSG A02Rik RIKEN cdna A02 gene E E-18 ENSMUSG Wscd1 WSC domain containing E E-18 ENSMUSG Ckap4 cytoskeleton-associated protein E E-18 ENSMUSG Cks1b CDC28 protein kinase 1b E E-18 ENSMUSG Hist1h1d histone cluster 1, H1d E E-18 ENSMUSG Kdelr3 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor E E-18 ENSMUSG Robo2 roundabout homolog 2 (Drosophila) E E-18 ENSMUSG Lgals1 lectin, galactose binding, soluble E E-17 ENSMUSG Tmem47 transmembrane protein E E-17 ENSMUSG Lims1 LIM and senescent cell antigen-like domains E E-17 ENSMUSG Kcne3 potassium voltage-gated channel, Isk-related subfamily, gene E E-17 ENSMUSG F2rl3 coagulation factor II (thrombin) receptor-like E E-17 ENSMUSG Pdzd2 PDZ domain containing E E-17 ENSMUSG Als2cl ALS2 C-terminal like E E-17 ENSMUSG Fn3k fructosamine 3 kinase E E-17 ENSMUSG Tacc3 transforming, acidic coiled-coil containing protein E E-17 ENSMUSG Ppic peptidylprolyl isomerase C E E-17 ENSMUSG Gper1 G protein-coupled estrogen receptor E E-17 ENSMUSG Exoc3l4 exocyst complex component 3-like E E-17 ENSMUSG Mcam melanoma cell adhesion molecule E E-17 ENSMUSG Smad9 SMAD family member E E-17 ENSMUSG Gtse1 G two S phase expressed protein E E-17 ENSMUSG Pfn1 profilin E E-17 ENSMUSG Suv39h2 suppressor of variegation 3-9 homolog 2 (Drosophila) E E-17 ENSMUSG Arsi arylsulfatase i E E-17 ENSMUSG Prrg3 proline rich Gla (G-carboxyglutamic acid) 3 (transmembrane) E E-17 ENSMUSG Stard8 START domain containing E E-17 ENSMUSG Vcan versican E E-17 ENSMUSG Galnt1 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase E E-17 ENSMUSG Klk8 kallikrein related-peptidase E E-17 ENSMUSG Marcksl1 MARCKS-like E E-17 ENSMUSG P07Rik RIKEN cdna P07 gene E E-17 ENSMUSG Lyrm9 LYR motif containing E E-17 ENSMUSG Nid2 nidogen E E-17 ENSMUSG Hrh2 histamine receptor H E E-17 ENSMUSG Csrp1 cysteine and glycine-rich protein E E-17 ENSMUSG Scamp5 secretory carrier membrane protein E E-17 ENSMUSG Pvrl2 poliovirus receptor-related E E-17 Page 3 of 84

12 ENSMUSG Ube2c ubiquitin-conjugating enzyme E2C E E-17 ENSMUSG Cyp1a1 cytochrome P450, family 1, subfamily a, polypeptide E E-17 ENSMUSG Gng2 guanine nucleotide binding protein (G protein), gamma E E-17 ENSMUSG Dgkb diacylglycerol kinase, beta E E-17 ENSMUSG Ighm immunoglobulin heavy constant mu E E-16 ENSMUSG Rassf10 Ras association (RalGDS/AF-6) domain family (N-terminal) member E E-16 ENSMUSG Tmem255a transmembrane protein 255A E E-16 ENSMUSG Aqp7 aquaporin E E-16 ENSMUSG Tnfrsf1b tumor necrosis factor receptor superfamily, member 1b E E-16 ENSMUSG Dclk1 doublecortin-like kinase E E-16 ENSMUSG Uhrf1bp1l UHRF1 (ICBP90) binding protein 1-like E E-16 ENSMUSG Adra2c adrenergic receptor, alpha 2c E E-16 ENSMUSG Dsp desmoplakin E E-16 ENSMUSG Gm694 predicted gene E E-16 ENSMUSG Ntng2 netrin G E E-16 ENSMUSG Gpsm2 G-protein signalling modulator 2 (AGS3-like, C. elegans) E E-16 ENSMUSG Upp1 uridine phosphorylase E E-16 ENSMUSG Cxcl12 chemokine (C-X-C motif) ligand E E-16 ENSMUSG Vmp1 vacuole membrane protein E E-16 ENSMUSG Gpr157 G protein-coupled receptor E E-16 ENSMUSG O22Rik RIKEN cdna O22 gene E E-16 ENSMUSG Epha4 Eph receptor A E E-16 ENSMUSG Fmo5 flavin containing monooxygenase E E-16 ENSMUSG Tmie transmembrane inner ear E E-16 ENSMUSG Prickle1 prickle homolog 1 (Drosophila) E E-16 ENSMUSG Cd200 CD200 antigen E E-16 ENSMUSG Tgtp1 T cell specific GTPase E E-16 ENSMUSG Cd44 CD44 antigen E E-16 ENSMUSG Pdk2 pyruvate dehydrogenase kinase, isoenzyme E E-16 ENSMUSG Rasgrp2 RAS, guanyl releasing protein E E-16 ENSMUSG Nes nestin E E-16 ENSMUSG Tnfrsf9 tumor necrosis factor receptor superfamily, member E E-16 ENSMUSG Stra6 stimulated by retinoic acid gene E E-16 ENSMUSG Procr protein C receptor, endothelial E E-16 ENSMUSG Slfn2 schlafen E E-16 ENSMUSG Eef1a2 eukaryotic translation elongation factor 1 alpha E E-16 ENSMUSG Car7 carbonic anhydrase E E-16 ENSMUSG Itgb1 integrin beta 1 (fibronectin receptor beta) E E-16 ENSMUSG Nod2 nucleotide-binding oligomerization domain containing E E-16 ENSMUSG Snn stannin E E-16 ENSMUSG Zfp52 zinc finger protein E E-16 ENSMUSG Tspan2 tetraspanin E E-16 ENSMUSG Aldh5a1 aldhehyde dehydrogenase family 5, subfamily A E E-16 ENSMUSG Cd274 CD274 antigen E E-16 ENSMUSG Smpdl3a sphingomyelin phosphodiesterase, acid-like 3A E E-16 ENSMUSG Sdcbp syndecan binding protein E E-16 ENSMUSG Akap6 A kinase (PRKA) anchor protein E E-16 ENSMUSG Cenpt centromere protein T E E-16 ENSMUSG Prr5l proline rich 5 like E E-16 ENSMUSG Pdgfa platelet derived growth factor, alpha E E-16 ENSMUSG Tagln2 transgelin E E-16 ENSMUSG Hist1h3b histone cluster 1, H3b E E-16 ENSMUSG Calu calumenin E E-16 ENSMUSG Pfkp phosphofructokinase, platelet E E-16 ENSMUSG Trpv1 transient receptor potential cation channel, subfamily V, member E E-15 ENSMUSG Kcnb1 potassium voltage gated channel, Shab-related subfamily, member E E-15 ENSMUSG Incenp inner centromere protein E E-15 ENSMUSG Rundc3b RUN domain containing 3B E E-15 ENSMUSG Prob1 proline rich basic protein E E-15 ENSMUSG Ablim2 actin-binding LIM protein E E-15 ENSMUSG Ppp1r12b protein phosphatase 1, regulatory (inhibitor) subunit 12B E E-15 ENSMUSG Myl2 myosin, light polypeptide 2, regulatory, cardiac, slow E E-15 ENSMUSG Nek6 NIMA (never in mitosis gene a)-related expressed kinase E E-15 ENSMUSG Myrip myosin VIIA and Rab interacting protein E E-15 ENSMUSG Mapk6 mitogen-activated protein kinase E E-15 ENSMUSG Olfr1396 olfactory receptor E E-15 ENSMUSG Cyp2d22 cytochrome P450, family 2, subfamily d, polypeptide E E-15 ENSMUSG Cyfip2 cytoplasmic FMR1 interacting protein E E-15 ENSMUSG Lif leukemia inhibitory factor E E-15 ENSMUSG Sdf2l1 stromal cell-derived factor 2-like E E-15 ENSMUSG L13Rik RIKEN cdna L13 gene E E-15 ENSMUSG Rad18 RAD18 homolog (S. cerevisiae) E E-15 ENSMUSG Il2ra interleukin 2 receptor, alpha chain E E-15 Page 4 of 84

13 ENSMUSG Rhbdl2 rhomboid, veinlet-like 2 (Drosophila) E E-15 ENSMUSG Ammecr1 Alport syndrome, mental retardation, midface hypoplasia and elliptocytosis chromosomal region gene E E-15 ENSMUSG Ntf3 neurotrophin E E-15 ENSMUSG Csgalnact1 chondroitin sulfate N-acetylgalactosaminyltransferase E E-15 ENSMUSG Lrp8 low density lipoprotein receptor-related protein 8, apolipoprotein e receptor E E-15 ENSMUSG Emp1 epithelial membrane protein E E-15 ENSMUSG Lrrc59 leucine rich repeat containing E E-15 ENSMUSG Cbx7 chromobox E E-15 ENSMUSG Alox12 arachidonate 12-lipoxygenase E E-15 ENSMUSG Mrvi1 MRV integration site E E-15 ENSMUSG F22Rik RIKEN cdna F22 gene E E-15 ENSMUSG H2-Ob histocompatibility 2, O region beta locus E E-15 ENSMUSG Hist1h1a histone cluster 1, H1a E E-15 ENSMUSG Fam83d family with sequence similarity 83, member D E E-15 ENSMUSG Gpr20 G protein-coupled receptor E E-15 ENSMUSG M07Rik RIKEN cdna M07 gene E E-15 ENSMUSG Ripk3 receptor-interacting serine-threonine kinase E E-15 ENSMUSG Lamc1 laminin, gamma E E-14 ENSMUSG Cdk1 cyclin-dependent kinase E E-14 ENSMUSG Armc2 armadillo repeat containing E E-14 ENSMUSG Pdlim1 PDZ and LIM domain 1 (elfin) E E-14 ENSMUSG Prelp proline arginine-rich end leucine-rich repeat E E-14 ENSMUSG Bambi-ps1 BMP and activin membrane-bound inhibitor, pseudogene (Xenopus laevis) E E-14 ENSMUSG Runx1t1 runt-related transcription factor 1; translocated to, 1 (cyclin D-related) E E-14 ENSMUSG Cdc20 cell division cycle E E-14 ENSMUSG Ctla2a cytotoxic T lymphocyte-associated protein 2 alpha E E-14 ENSMUSG Eno3 enolase 3, beta muscle E E-14 ENSMUSG Nov nephroblastoma overexpressed gene E E-14 ENSMUSG Kif2c kinesin family member 2C E E-14 ENSMUSG Ly6c1 lymphocyte antigen 6 complex, locus C E E-14 ENSMUSG Nek2 NIMA (never in mitosis gene a)-related expressed kinase E E-14 ENSMUSG Pisd phosphatidylserine decarboxylase E E-14 ENSMUSG Atp10d ATPase, class V, type 10D E E-14 ENSMUSG Syne1 spectrin repeat containing, nuclear envelope E E-14 ENSMUSG Cmpk1 cytidine monophosphate (UMP-CMP) kinase E E-14 ENSMUSG Trim65 tripartite motif-containing E E-14 ENSMUSG Pdia6 protein disulfide isomerase associated E E-14 ENSMUSG Lama3 laminin, alpha E E-14 ENSMUSG Slc25a23 solute carrier family 25 (mitochondrial carrier; phosphate carrier), member E E-14 ENSMUSG Slc2a4 solute carrier family 2 (facilitated glucose transporter), member E E-14 ENSMUSG Spred3 sprouty-related, EVH1 domain containing E E-14 ENSMUSG Ptprg protein tyrosine phosphatase, receptor type, G E E-14 ENSMUSG Clic1 chloride intracellular channel E E-14 ENSMUSG Kifc1 kinesin family member C E E-14 ENSMUSG Tcea3 transcription elongation factor A (SII), E E-14 ENSMUSG Edem1 ER degradation enhancer, mannosidase alpha-like E E-14 ENSMUSG Troap trophinin associated protein E E-14 ENSMUSG Irf8 interferon regulatory factor E E-14 ENSMUSG Ctla2b cytotoxic T lymphocyte-associated protein 2 beta E E-14 ENSMUSG Kif22 kinesin family member E E-14 ENSMUSG Flrt2 fibronectin leucine rich transmembrane protein E E-14 ENSMUSG Aspa aspartoacylase E E-14 ENSMUSG Pnkd paroxysmal nonkinesiogenic dyskinesia E E-14 ENSMUSG Bean1 brain expressed, associated with Nedd4, E E-14 ENSMUSG Sun1 Sad1 and UNC84 domain containing E E-14 ENSMUSG Kcnmb4 potassium large conductance calcium-activated channel, subfamily M, beta member E E-14 ENSMUSG Ret ret proto-oncogene E E-14 ENSMUSG Tuba1a tubulin, alpha 1A E E-14 ENSMUSG Col5a2 collagen, type V, alpha E E-14 ENSMUSG Fam78b family with sequence similarity 78, member B E E-14 ENSMUSG Tex2 testis expressed gene E E-14 ENSMUSG Synj2 synaptojanin E E-14 ENSMUSG Psmd14 proteasome (prosome, macropain) 26S subunit, non-atpase, E E-13 ENSMUSG Vash1 vasohibin E E-13 ENSMUSG Hcls1 hematopoietic cell specific Lyn substrate E E-13 ENSMUSG Arrdc3 arrestin domain containing E E-13 ENSMUSG Stil Scl/Tal1 interrupting locus E E-13 ENSMUSG Cfl1 cofilin 1, non-muscle E E-13 ENSMUSG Spats2 spermatogenesis associated, serine-rich E E-13 ENSMUSG Cxcl10 chemokine (C-X-C motif) ligand E E-13 ENSMUSG Samsn1 SAM domain, SH3 domain and nuclear localization signals, E E-13 ENSMUSG Pno1 partner of NOB1 homolog (S. cerevisiae) E E-13 ENSMUSG Manf mesencephalic astrocyte-derived neurotrophic factor E E-13 Page 5 of 84

14 ENSMUSG Grip2 glutamate receptor interacting protein E E-13 ENSMUSG Rab6b RAB6B, member RAS oncogene family E E-13 ENSMUSG Il1a interleukin 1 alpha E E-13 ENSMUSG Bora bora, aurora kinase A activator E E-13 ENSMUSG Lxn latexin E E-13 ENSMUSG Tmem38a transmembrane protein 38A E E-13 ENSMUSG Hif1a hypoxia inducible factor 1, alpha subunit E E-13 ENSMUSG Ctsf cathepsin F E E-13 ENSMUSG Kcnn3 potassium intermediate/small conductance calcium-activated channel, subfamily N, member E E-13 ENSMUSG Hn1l hematological and neurological expressed 1-like E E-13 ENSMUSG Plce1 phospholipase C, epsilon E E-13 ENSMUSG Cacna1g calcium channel, voltage-dependent, T type, alpha 1G subunit E E-13 ENSMUSG Atp7a ATPase, Cu++ transporting, alpha polypeptide E E-13 ENSMUSG Ppfibp2 PTPRF interacting protein, binding protein 2 (liprin beta 2) E E-13 ENSMUSG Tdpx-ps1 thioredoxin peroxidase, pseudogene 1 Inf 3.74E E-13 ENSMUSG Akap7 A kinase (PRKA) anchor protein E E-13 ENSMUSG Psmd8 proteasome (prosome, macropain) 26S subunit, non-atpase, E E-13 ENSMUSG Gm9917 predicted gene E E-13 ENSMUSG Bmp2k BMP2 inducible kinase E E-13 ENSMUSG Adamtsl3 ADAMTS-like E E-13 ENSMUSG Myadm myeloid-associated differentiation marker E E-13 ENSMUSG Ppm1l protein phosphatase 1 (formerly 2C)-like E E-13 ENSMUSG Tspan18 tetraspanin E E-13 ENSMUSG Soat1 sterol O-acyltransferase E E-13 ENSMUSG AI expressed sequence AI E E-13 ENSMUSG Anxa2 annexin A E E-13 ENSMUSG Gm22973 predicted gene, E E-13 ENSMUSG Trpc3 transient receptor potential cation channel, subfamily C, member E E-13 ENSMUSG Sned1 sushi, nidogen and EGF-like domains E E-13 ENSMUSG Cnksr3 Cnksr family member E E-13 ENSMUSG Fam49a family with sequence similarity 49, member A E E-13 ENSMUSG Cyyr1 cysteine and tyrosine-rich protein E E-13 ENSMUSG Crhbp corticotropin releasing hormone binding protein E E-13 ENSMUSG Cdca8 cell division cycle associated E E-13 ENSMUSG Phf1 PHD finger protein E E-13 ENSMUSG Tmc5 transmembrane channel-like gene family E E-13 ENSMUSG Acsl4 acyl-coa synthetase long-chain family member E E-13 ENSMUSG Col4a1 collagen, type IV, alpha E E-13 ENSMUSG Cst3 cystatin C E E-13 ENSMUSG Pkig protein kinase inhibitor, gamma E E-13 ENSMUSG Kif20a kinesin family member 20A E E-13 ENSMUSG Acot7 acyl-coa thioesterase E E-13 ENSMUSG Klk10 kallikrein related-peptidase E E-13 ENSMUSG Timp1 tissue inhibitor of metalloproteinase E E-13 ENSMUSG Prkg2 protein kinase, cgmp-dependent, type II E E-13 ENSMUSG Wdr62 WD repeat domain E E-13 ENSMUSG Nudt12 nudix (nucleoside diphosphate linked moiety X)-type motif E E-13 ENSMUSG Mlkl mixed lineage kinase domain-like E E-13 ENSMUSG Tnfrsf23 tumor necrosis factor receptor superfamily, member E E-13 ENSMUSG Oas2 2'-5' oligoadenylate synthetase E E-13 ENSMUSG Knstrn kinetochore-localized astrin/spag5 binding E E-13 ENSMUSG Psmc4 proteasome (prosome, macropain) 26S subunit, ATPase, E E-13 ENSMUSG Rnf122 ring finger protein E E-13 ENSMUSG Fam117a family with sequence similarity 117, member A E E-13 ENSMUSG Fhl3 four and a half LIM domains E E-13 ENSMUSG Slc39a6 solute carrier family 39 (metal ion transporter), member E E-13 ENSMUSG C19Rik RIKEN cdna C19 gene E E-13 ENSMUSG Iqgap3 IQ motif containing GTPase activating protein E E-13 ENSMUSG Myh14 myosin, heavy polypeptide E E-13 ENSMUSG Slfn3 schlafen E E-13 ENSMUSG Gucy1a3 guanylate cyclase 1, soluble, alpha E E-13 ENSMUSG Gars glycyl-trna synthetase E E-13 ENSMUSG Pcsk6 proprotein convertase subtilisin/kexin type E E-12 ENSMUSG Mmp14 matrix metallopeptidase 14 (membrane-inserted) E E-12 ENSMUSG Mill2 MHC I like leukocyte E E-12 ENSMUSG Ddias DNA damage-induced apoptosis suppressor E E-12 ENSMUSG Nxpe4 neurexophilin and PC-esterase domain family, member E E-12 ENSMUSG Ppap2c phosphatidic acid phosphatase type 2C E E-12 ENSMUSG Fam167a family with sequence similarity 167, member A E E-12 ENSMUSG Rhoc ras homolog gene family, member C E E-12 ENSMUSG Actr3 ARP3 actin-related protein E E-12 ENSMUSG P21Rik RIKEN cdna P21 gene E E-12 ENSMUSG Pln phospholamban E E-12 Page 6 of 84

15 ENSMUSG Zmat3 zinc finger matrin type E E-12 ENSMUSG Tnfaip8l1 tumor necrosis factor, alpha-induced protein 8-like E E-12 ENSMUSG Erbb3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian) E E-12 ENSMUSG Garem GRB2 associated, regulator of MAPK E E-12 ENSMUSG Cenpq centromere protein Q E E-12 ENSMUSG Arpc5 actin related protein 2/3 complex, subunit E E-12 ENSMUSG Tnfsf10 tumor necrosis factor (ligand) superfamily, member E E-12 ENSMUSG Tcf23 transcription factor E E-12 ENSMUSG Memo1 mediator of cell motility E E-12 ENSMUSG Cyp51 cytochrome P450, family E E-12 ENSMUSG Sema3a sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A E E-12 ENSMUSG C1qtnf6 C1q and tumor necrosis factor related protein E E-12 ENSMUSG Synpo2 synaptopodin E E-12 ENSMUSG Cd22 CD22 antigen E E-12 ENSMUSG Fam20a family with sequence similarity 20, member A E E-12 ENSMUSG Nrbp2 nuclear receptor binding protein E E-12 ENSMUSG Dgat2 diacylglycerol O-acyltransferase E E-12 ENSMUSG Wfdc1 WAP four-disulfide core domain E E-12 ENSMUSG Fgfr3 fibroblast growth factor receptor E E-12 ENSMUSG Nin ninein E E-12 ENSMUSG Svip small VCP/p97-interacting protein E E-12 ENSMUSG Asb2 ankyrin repeat and SOCS box-containing E E-12 ENSMUSG Zrsr1 zinc finger (CCCH type), RNA binding motif and serine/arginine rich E E-12 ENSMUSG Ldlr low density lipoprotein receptor E E-12 ENSMUSG Mtus2 microtubule associated tumor suppressor candidate E E-12 ENSMUSG Mgat5 mannoside acetylglucosaminyltransferase E E-12 ENSMUSG Asns asparagine synthetase E E-12 ENSMUSG Thrb thyroid hormone receptor beta E E-12 ENSMUSG Nudt4 nudix (nucleoside diphosphate linked moiety X)-type motif E E-12 ENSMUSG Dnase2a deoxyribonuclease II alpha E E-12 ENSMUSG Thyn1 thymocyte nuclear protein E E-12 ENSMUSG Lyrm5 LYR motif containing E E-12 ENSMUSG Igfbp7 insulin-like growth factor binding protein E E-12 ENSMUSG Ip6k3 inositol hexaphosphate kinase E E-12 ENSMUSG Chd7 chromodomain helicase DNA binding protein E E-12 ENSMUSG Hist1h1b histone cluster 1, H1b E E-12 ENSMUSG Cmah cytidine monophospho-n-acetylneuraminic acid hydroxylase E E-12 ENSMUSG Txn1 thioredoxin E E-12 ENSMUSG Pif1 PIF1 5'-to-3' DNA helicase homolog (S. cerevisiae) E E-12 ENSMUSG Gm13861 predicted gene E E-12 ENSMUSG Ecm2 extracellular matrix protein 2, female organ and adipocyte specific E E-12 ENSMUSG Itga9 integrin alpha E E-12 ENSMUSG Fabp5 fatty acid binding protein 5, epidermal E E-12 ENSMUSG Cd40 CD40 antigen E E-12 ENSMUSG Naa50 N(alpha)-acetyltransferase 50, NatE catalytic subunit E E-12 ENSMUSG Calr3 calreticulin E E-12 ENSMUSG Tmem140 transmembrane protein E E-12 ENSMUSG Ephx1 epoxide hydrolase 1, microsomal E E-12 ENSMUSG RP24-422C E E-12 ENSMUSG Cda cytidine deaminase E E-12 ENSMUSG Plcb1 phospholipase C, beta E E-12 ENSMUSG Tada1 transcriptional adaptor E E-12 ENSMUSG Dnah6 dynein, axonemal, heavy chain E E-12 ENSMUSG AA expressed sequence AA E E-12 ENSMUSG Smad1 SMAD family member E E-12 ENSMUSG Bmx BMX non-receptor tyrosine kinase E E-12 ENSMUSG Asb10 ankyrin repeat and SOCS box-containing E E-12 ENSMUSG Ncaph non-smc condensin I complex, subunit H E E-12 ENSMUSG Abracl ABRA C-terminal like E E-12 ENSMUSG Nipal3 NIPA-like domain containing E E-12 ENSMUSG Klhl38 kelch-like E E-12 ENSMUSG Aurka aurora kinase A E E-12 ENSMUSG Asb18 ankyrin repeat and SOCS box-containing E E-12 ENSMUSG Kif18b kinesin family member 18B E E-12 ENSMUSG Tubb2b tubulin, beta 2B class IIB E E-11 ENSMUSG Pgm2l1 phosphoglucomutase 2-like E E-11 ENSMUSG Crmp1 collapsin response mediator protein E E-11 ENSMUSG Sstr4 somatostatin receptor E E-11 ENSMUSG Setbp1 SET binding protein E E-11 ENSMUSG Olfr78 olfactory receptor E E-11 ENSMUSG Hspa5 heat shock protein E E-11 ENSMUSG Mgat4a mannoside acetylglucosaminyltransferase 4, isoenzyme A E E-11 ENSMUSG Gm16340 predicted gene E E-11 Page 7 of 84