Supplemental Material

Small Intestinal Stem Cell Identity Is Maintained with Functional Paneth Cells in Heterotopically Grafted Epithelium onto Colon

Masayoshi Fukuda, Tomohiro Mizutani, Wakana Mochizuki, Taichi Matsumoto, Kengo Nozaki, Yuriko Sakamaki, Shizuko Ichinose, Yukinori Okada, Toshihiro Tanaka, Mamoru Watanabe, Tetsuya Nakamura

Supplemental Materials and Methods

Supplemental Figures and Figure Legends: Supplemental Figure S1 Colonic epithelial injury model used in the present study Supplemental Figure S2 Freshly isolated SI epithelia also maintain SI phenotype when grafted Supplemental Figure S3 Subepithelium of the graft contains multiple cellular components of recipient origin Supplemental Figure S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation Supplemental Figure S5 Laser capture microdissection and following microarray analysis

Supplemental Table S1 List of differentially expressed between the graft and normal colonic epithelium Supplemental Materials and Methods

Stereomicroscopy, Histology, and Immunohistochemistry

Whole distal colons of recipients and their fluorescence were imaged using a fluorescence stereomicroscope system MVX10 (Olympus). For histology and immunohistochemistry, recipient colons were fixed overnight at 4˚C in 4% paraformaldehyde, sequentially dehydrated in 10, 15 and 20% sucrose in PBS, and embedded in OCT compound (Tissue Tek). Frozen sections of 6-mm thickness were cut, mounted on slide glasses, and examined for their fluorescent signals in order to determine whether the section contains EGFP+ grafts. Sections containing

EGFP+ grafts were subjected to H&E staining or immunohistochemistry. The followings were used as antibodies specific for each : Cdh (Cell Signaling); CA2 (Santa Cruz); CDX2 (Bio Genex); Ki67 (Dako Cytomation);

MUC2 (Santa Cruz); ChgA (ImmunoStar); sucrose isomaltase (Santa Cruz); Lysozyme (DAKO); CD3 (BD); F4/80

(Abcam); and CD31 (Abcam). In all immunofluorescent experiments, nuclei were counterstained with DAPI.

Sections obtained from some tissue blocks showed obvious but weak endogenous EGFP signals presumably because of the variation in the rate and quality of fixation. In those cases, we performed immunofluorescence staining of adjacent sections with anti GFP antibody (Invitrogen) and presented them to show the EGFP fluorescence as a reference. Fluorescent images of sections were acquired using a DeltaVision system (Applied

Precision) where a fluorescent microscope IX-71 (Olympus) with objectives UplansApo 10x 0.4NA or UplansApo

20x 0.75NA (Olympus) is incorporated. If necessary, image processing was carried out using Adobe Photoshop

Elements 7.0 software. For quantification of cellular components, sections of proximal, middle, distal SI, colon, and EGFP+ grafts were subjected to immunohistochemistry for ChgA, MUC2, or Lysozyme. Thirty crypt-villus units or crypt units (colon), originating from 3 independent recipients or normal controls, were analyzed. The numbers of ChgA+, MUC2+, or Lysozyme+ cells were counted and presented as mean cell counts per crypt-villus or crypt unit. Statistical significance was determined by Student’s t test (p < 0.05).

Laser Capture Microdissection

Recipient mice at 4 weeks post-transplantation and C57BL normal control mice were sacrificed. The graft-containing recipients’ tissues and control colon tissues were quickly washed in PBS, embedded in OCT compound (Tissue Tek), rapidly frozen, and stored. For recipients’ colon experiments, we searched and located the plane of specimens so that the section would contain EGFP+ grafts. From there onwards, serial cryosections

(10-mm thickness) of more than 30 slices were made. Fluorescent images of a part of these slices (1 every 7 sections) were acquired, and they were used as references for dissecting the EGFP+ epithelia from sections located in between. Likewise, we also made ~ 30 sections from the distal colon of control mice. Sections were stained with cresyl violet solution and Arcturus HistGene Staining Solution (Applied Biosystems). Grafted epithelia in recipients or control colonic epithelia were microdissected using Micro Laser System MBIII

(PALM/ZEISS). Dissected epithelia obtained from serial sections (~ 30 sections) of each specimen were combined and then total RNA extraction was performed by using RNeasy Plus Micro Kit (QUIAGEN). After calculating RNA Integrity Number (RIN), we subjected one graft sample and one control colon sample, which showed the highest RIN value, for the following expression analysis.

Microarray and Data Analysis cDNA synthesis, amplification and labeling were performed with Ovation Pico WTA System V2 and Encore Biotin

Module (Nugen). Microarray hybridization was carried out onto GeneChip Mouse Gene 1.0 ST Array (Affymetrix).

The arrays were scanned with GeneChip Scanner 3000 7G (Affymetrix). Data files were imported to GeneSpring

GX 12.5 software (Agilent), and its Robust Multichip Average algorithm 16 (RMA16) was used for data normalization. The bottom 20% of genes with the lowest expression levels was excluded from subsequent analysis. Multiple probesets on Affymetrix microarrays are associated with transcript clusters. Transcript clusters that showed > 3-fold differences in expression values between two samples are presented. For a gene or a group of genes to which values are given by multiple transcript clusters, averages of the values for those clusters were used for assessing fold enrichment. Detailed information on the probeset and transcript cluster grouping is available on the NetAffyx site (http://www.affymetrix.com/estore/index.jsp) provided by the manufacturer. Fukuda et al., Supplemental Figure S1

A

B Day 2 Day 2

Day 14 Day 14

Day 28 Day 28

Supplemental Fig. S1 Colonic epithelial injury model used in the present study. (A) Devices used to generate distal colonic epithelial injury. Thin catheter equipped with two small rubber balloons used for topical EDTA treatment (left). After the EDTA treatment, balloons were deflated and the catheter was removed out of the colon. Epithelial abrasion was performed by using an electric brush (right), giving its rotational movement to scratch the colonic luminal surface. (B) Time course change of the tissue damage during recovery. Stereoscopic images (left) at Day 2, 14, 28 after injury are presented with their longitudinal sections (right). H&E-stained sections are shown with anal side on the left and oral side on the right. High-power views of areas in dotted rectangles are shown at the bottom. Scale bars show 1 mm. Fukuda et al., Supplemental Figure S2

A A´

Lysozyme EGFP

B B´ B´´

CDX2 CA2 EGFP

Supplemental Fig. S2 Freshly isolated SI epithelia also maintain SI phenotype when grafted. SI epithelia were isolated from EGFP transgenic mice and transplanted immediately into recipients without culturing. Recipient colons at 2 weeks post-transplantation were analyzed. (A) immunostaining for Lysozyme (A) and endogenous EGFP fluorescence (A´) of the same section. (B) Section double stained with CDX2 (B) and CA2 (B´) is presented with the endogenous EGFP fluorescence of the same section (B´´). Note that the antigen retrieval procedure before immunostaining completely clears endogenous EGFP signals.Images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm. Fukuda et al., Supplemental Figure S3

A CD3 A´ EGFP

B F4/80 B´ EGFP

C CD31 C´ EGFP

Supplemental Fig. S3 Subepithelium of the graft contains multiple cellular components of recipient origin. Immunohistochemical analysis of recipient colons at 4 weeks post-transplantation. Tissue sections were immunostained with anti-CD3 (A), anti-F4/80 (B), or anti-CD31 (C) antibody to detect T cells, monocyte/macrophage lineage of cells, and blood vessel endothelial cells, respectively. EGFP fluorescence of the same sections (A´, B´, and C´) is also shown. Arrowheads point to positively-stained cells residing within the subepithelia entirely surrounded by EGFP+ epithelia. Images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm. Fukuda et al., Supplemental Figure S4

A D

A´ C´ Lysozyme

CA2 B EGFP D´

SIase C Lysozyme D´´

EGFP

E CDX2 F Ki67 G H

MUC2 ChgA E´ F´ G´ H´

EGFP EGFP EGFP EGFP

Supplemental Fig. S4 Multi-differentiation and self-renewal capabilities of grafted cells at 4 months post-transplantation. (A) H&E staining of a section obtained from a recipient colon at 4 months (18 weeks) post-transplantation. (B,C) EGFP fluorescence (B) of the section adjacent to the one shown in (A) and its immunostaining for Lysozyme (C). High-power views of areas in dotted boxes in (A) and (C) are shown as (A´) and (C´), respectively. (D) Section double stained with CA2 (D) and sucrase isomaltase (SIase; D´) is presented with the endogenous EGFP fluorescence of the same section (D´´). Note that the antigen retrieval procedure before immunostaining completely clears endogenous EGFP signals. (E-H) Immunofluorescence for CDX2 (E), Ki67 (F), MUC2 (G), and ChgA (H). Arrowheads point to ChgA+ cells. Images of endogenous EGFP fluorescence of the same sections (E´, F´, G´) or an adjacent section (H´) are shown. Fluorescent images are shown with DAPI staining. Dotted lines indicate borders between EGFP+ and EGFP- epithelia. Scale bars; 100 μm. Fukuda et al., Supplemental Figure S5

Graft Control Colon

EGFP Pre Dissection

Pre Dissection Mesenchyme elimination

Post Dissection Post Dissection

RNA Extraction

cDNA Amplification

Microarray Analysis

Supplemental Fig. S5 Laser capture microdissection and following microarray analysis. The graft-containing tissues and control colon tissues were obtained from recipient mice (4 weeks post-transplantation) and normal syngeneic mice, respectively. To obtain donor-derived epithelia from recipients’ colon, serial sections were prepared per sample so that all sections contain EGFP+ epithelia. Every 6th section was used to detect EGFP+ signals as references for dissecting the EGFP+ epithelia from other sections located in between. Microdissected epithelia from multiple sections of each sample were combined and then total RNA was extracted. RNA was converted to cDNA, amplified, labeled, and used for GeneChip hybridization. Scale bars show 100 μm. Supplemental Table S1. List of genes differentially expressed between the graft and normal colonic epithelium. Listed in this table are the transcript clusters (See Experimental Procedures) that showed > 3-fold differences in expression values between two samples. For a gene or a group of genes to which values are given by multiple transcript clusters, the average of values for those clusters was used for assessing fold enrichment and presented.

List of genes enriched in Graft No Affimetrix Transcripts Cluster ID Gene Symbol Gene Description Fold Change Graft/Colon 1 10550131 Pla2g4c A2, group IVC (cytosolic, calcium-independent) 129.0492 2 10419575 Ang4 , A family, member 4 111.4434 Defa24 defensin, alpha, 24 3 10570735 102.2967 Defa6 defensin, alpha, 6 Defa23 defensin, alpha, 23 Defa-rs7 defensin, alpha, related sequence 7 10570656 4 Defa17 defensin, alpha, 17 73.6512 10570676 Defa3 defensin, alpha, 3 Gm15284 predicted gene 15284 Gm15284 predicted gene 15284 Defa17 defensin, alpha, 17 Defa3 defensin, alpha, 3 5 10570668 Defa24 defensin, alpha, 24 71.0562 Defa6 defensin, alpha, 6 Defa23 defensin, alpha, 23 Defa-rs7 defensin, alpha, related sequence 7 Defa17 defensin, alpha, 17 Defa3 defensin, alpha, 3 Gm15284 predicted gene 15284 10570690 6 Defa23 defensin, alpha, 23 60.7345 10570720 Defa-rs7 defensin, alpha, related sequence 7 Defa13 defensin, alpha, 13 Defa15 defensin, alpha, 15 7 10501026 Chi3l4 chitinase 3-like 4 60.5800 8 10538965 Fabp1 fatty acid binding protein 1, liver 50.7614 9 10356367 Alpi alkaline , intestinal 48.5584 10585010 10 Apoa4 apolipoprotein A-IV 48.3594 10593174 11 10436087 Retnlb resistin like beta 47.6103 12 10585005 Apoa1 apolipoprotein A-I 46.7744 13 10498659 Sis sucrase isomaltase (alpha-glucosidase) 43.7697 14 10558590 Prap1 proline-rich acidic protein 1 42.0095 15 10416689 Olfm4 olfactomedin 4 34.0355 16 10502613 AI747448 expressed sequence AI747448 31.7306 17 10464328 Pnliprp2 pancreatic -related protein 2 29.7698 Itln1 intelectin 1 (galactofuranose binding) 18 10360149 29.4274 Itlnb intelectin b 19 10347117 Cps1 carbamoyl-phosphate synthetase 1 25.0372 20 10430425 Lgals2 lectin, galactose-binding, soluble 2 22.8981 Defa-ps1 defensin, alpha, pseudogene 1 Defa25 defensin, alpha, 25 Defa17 defensin, alpha, 17 Defa3 defensin, alpha, 3 Defa23 defensin, alpha, 23 21 10570683 20.8023 Defa-rs7 defensin, alpha, related sequence 7 Gm15299 predicted pseudogene 15299 Defa2 defensin, alpha, 2 Gm15292 predicted gene 15292 Defa26 defensin, alpha, 26 22 10372652 Lyz1 lysozyme 1 19.8162 Defa25 defensin, alpha, 25 Gm15299 predicted pseudogene 15299 23 10570700 Gm6696 predicted gene 6696 19.0097 Defa-ps1 defensin, alpha, pseudogene 1 Defa23 defensin, alpha, 23 24 10583133 Mmp7 matrix metallopeptidase 7 18.6278 25 10402708 Ckb creatine kinase, brain 18.1232 26 10531407 Cxcl9 chemokine (C-X-C motif) ligand 9 17.0413 27 10384102 Npc1l1 NPC1-like 1 16.1600 28 10502375 Mttp microsomal triglyceride transfer protein 14.5011 10595480 29 Me1 malic 1, NADP(+)-dependent, cytosolic 13.5514 10405753 30 10456005 Cd74 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) 12.8037 31 10368317 Enpp3 ectonucleotide pyrophosphatase/ 3 12.5226 32 10549154 Gm766 predicted gene 766 11.7360 33 10543411 Slc13a1 solute carrier family 13 (sodium/sulfate symporters), member 1 11.6542 34 10438567 2310042E22Rik RIKEN cDNA 2310042E22 gene 11.5431 35 10444291 H2-Ab1 histocompatibility 2, class II antigen A, beta 1 11.4623 36 10489391 Ada adenosine deaminase 11.3608 37 10475448 Duoxa2 dual oxidase maturation factor 2 10.9206 38 10445112 Ubd ubiquitin D 10.4157 39 10545208 Gm189 predicted gene 189 10.3052 40 10369567 Hkdc1 hexokinase domain containing 1 9.7970 Arg2 arginase type II 41 10396831 9.7368 Vti1b vesicle transport through interaction with t-SNAREs 1B homolog Defa25 defensin, alpha, 25 Gm6696 predicted gene 6696 42 10570663 Defa-ps1 defensin, alpha, pseudogene 1 9.1532 Gm15299 predicted pseudogene 15299 Defa23 defensin, alpha, 23 43 10386197 2210407C18Rik RIKEN cDNA 2210407C18 gene 8.8244 44 10552193 Slc7a9 solute carrier family 7 (cationic amino acid transporter, y+ system), member 9 8.7444 Defa20 defensin, alpha, 20 10570711 45 Gm15308 predicted gene 15308 8.7175 10570706 Defa3 defensin, alpha, 3 46 10407337 Hcn1 hyperpolarization-activated, cyclic nucleotide-gated K+ 1 8.6979 47 10486956 Duox2 dual oxidase 2 8.6024 48 10372557 Gm239 predicted gene 239 8.4779 Tgtp1 T-cell specific GTPase 1 10385518 49 Tgtp2 T-cell specific GTPase 2 8.4646 10385533 Gm12185 predicted gene 12185 Defa26 defensin, alpha, 26 50 10570726 Defa23 defensin, alpha, 23 7.9827 Defa-rs7 defensin, alpha, related sequence 7 51 10384044 Myl7 myosin, light polypeptide 7, regulatory 7.9675 52 10539200 Reg1 regenerating islet-derived 1 7.9573 53 10405587 Tgfbi transforming growth factor, beta induced 7.8007 54 10425037 Apol10a apolipoprotein L 10a 7.6631 55 10588043 Rbp2 retinol binding protein 2, cellular 7.4315 56 10539179 Reg3b regenerating islet-derived 3 beta 6.9385 57 10419563 Rnase1 ribonuclease, RNase A family, 1 (pancreatic) 6.8738 58 10380581 Gip gastric inhibitory polypeptide 6.7543 59 10394394 Apob apolipoprotein B 6.5556 60 10527638 Alox5ap arachidonate 5-lipoxygenase activating protein 6.2527 10489721 10363561 10427389 10565598 10424377 10422247 10568534 10528205 10603549 10500527 10410927 10581009 10478746 10589974 10528165 10541129 10543029 10345436 10395275 10346222 10457667 10484355 10607429 10483161 10497327 61 LOC280487 pol polyprotein 6.1985 10586863 10468487 10469127 10374352 10374183 10356762 10540531 10527425 10603803 10357298 10476399 10522742 10363005 10574432 10574434 10496167 10582983 10512463 10496336 10359642 10448230 10416698 10485355 10416696 10375324 62 10377841 Tm4sf5 transmembrane 4 superfamily member 5 6.1292 Defa5 defensin, alpha, 5 Gm15315 predicted gene 15315 63 10570693 Gm14850 predicted gene 14850 6.1097 Gm10104 predicted gene 10104 Gm15293 predicted gene 15293 64 10420747 Gata4 GATA binding protein 4 6.0913 65 10455961 Iigp1 interferon inducible GTPase 1 6.0731 66 10388902 Lgals9 lectin, galactose binding, soluble 9 6.0658 Gm1943 WD repeat domain 70 pseudogene 67 10581575 6.0203 Wdr70 Gm15315 predicted gene 15315 Defa5 defensin, alpha, 5 Gm14850 predicted gene 14850 68 10570732 5.9227 Gm10104 predicted gene 10104 Gm15293 predicted gene 15293 Defa4 defensin, alpha, 4 10403034 69 LOC100046275 ig heavy chain V-II region SESS-like 5.6599 10403063 70 10603066 Ace2 angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 5.6561 71 10358399 Rgs13 regulator of G-protein signaling 13 5.6483 Gm10104 predicted gene 10104 Gm14850 predicted gene 14850 Defa5 defensin, alpha, 5 72 10570660 5.5796 Gm15315 predicted gene 15315 Gm15293 predicted gene 15293 Defa4 defensin, alpha, 4 Gm14850 predicted gene 14850 Gm10104 predicted gene 10104 Defa5 defensin, alpha, 5 73 10570717 5.4712 Gm15315 predicted gene 15315 Gm15293 predicted gene 15293 Defa4 defensin, alpha, 4 74 10363372 Pla2g12b , group XIIB 5.4279 Chn2 chimerin (chimaerin) 2 75 10538356 5.4086 9130019P16Rik RIKEN cDNA 9130019P16 gene 76 10546929 Cidec cell death-inducing DFFA-like effector c 5.3987 77 10570018 Tnfsf13b tumor necrosis factor (ligand) superfamily, member 13b 5.2794 78 10546685 Eif4e3 eukaryotic translation initiation factor 4E member 3 5.2484 79 10543319 Fam3c family with sequence similarity 3, member C 5.1963 Defa22 defensin, alpha, 22 80 10570671 5.1204 Defa21 defensin, alpha, 21 81 10457475 Abhd3 abhydrolase domain containing 3 4.9802 82 10444298 H2-Eb1 histocompatibility 2, class II antigen E beta 4.9636 83 10538871 Gm4964 predicted gene 4964 4.8887 Gm6696 predicted gene 6696 Defa25 defensin, alpha, 25 84 10570703 4.8801 Gm15299 predicted pseudogene 15299 Defa-ps1 defensin, alpha, pseudogene 1 85 10489759 Sulf2 2 4.8775 Defa21 defensin, alpha, 21 86 10570651 4.8610 Defa22 defensin, alpha, 22 87 10542857 Far2 fatty acyl CoA reductase 2 4.8578 88 10362674 Rnu3a U3A small nuclear RNA 4.8475 89 10560719 2210010C17Rik RIKEN cDNA 2210010C17 gene 4.8298 90 10449467 Clps colipase, pancreatic 4.8114 91 10589166 Slc26a6 solute carrier family 26, member 6 4.7743 92 10607602 Ppef1 with EF hand calcium-binding domain 1 4.7565 93 10408629 1300014I06Rik RIKEN cDNA 1300014I06 gene 4.7448 94 10424430 Gsdmcl-ps gasdermin C-like, pseudogene 4.7265 10354649 95 Pgap1 post-GPI attachment to 1 4.6861 10354647 96 10545425 Sh2d6 SH2 domain containing 6 4.6303 B4galnt1 beta-1,4-N-acetyl-galactosaminyl 1 97 10366848 4.5800 Slc26a10 solute carrier family 26, member 10 98 10444229 H2-DMa histocompatibility 2, class II, locus DMa 4.5683 99 10465831 5730408K05Rik RIKEN cDNA 5730408K05 gene 4.5082 100 10593473 1810046K07Rik RIKEN cDNA 1810046K07 gene 4.5000 101 10429926 Dgat1 diacylglycerol O-acyltransferase 1 4.4767 102 10511429 Car8 carbonic anhydrase 8 4.4738 Defa25 defensin, alpha, 25 103 10570723 Gm6696 predicted gene 6696 4.4495 Gm15299 predicted pseudogene 15299 104 10569962 Ccl25 chemokine (C-C motif) ligand 25 4.4339 105 10348451 Cxcr7 chemokine (C-X-C motif) receptor 7 4.4174 106 10422760 Fyb FYN binding protein 4.4159 107 10428955 Gsdmc2 gasdermin C2 4.3435 108 10495820 Fabp2 fatty acid binding protein 2, intestinal 4.3159 109 10355403 Fn1 fibronectin 1 4.3106 110 10572271 Tm6sf2 transmembrane 6 superfamily member 2 4.2767 111 10408613 Tubb2b tubulin, beta 2B 4.2559 112 10419198 Ero1l ERO1-like (S. cerevisiae) 4.2502 113 10527565 Pdx1 pancreatic and duodenal homeobox 1 4.2063 114 10475487 Slc28a2 solute carrier family 28 (sodium-coupled nucleoside transporter), member 2 4.1907 115 10498710 Bche 4.1873 116 10383930 Gm10278 predicted gene 10278 4.1861 117 10446656 Lpin2 lipin 2 4.1630 Abpd androgen binding protein delta 118 10562291 4.1343 Gm9138 predicted gene 9138 119 10450154 H2-Aa histocompatibility 2, class II antigen A, alpha 4.0963 120 10371054 Creb3l3 cAMP responsive element binding protein 3-like 3 4.0921 Defa-rs1 defensin, alpha, related sequence 1 Gm14851 predicted gene 14851 121 10577426 Gm7849 predicted gene 7849 4.0639 Gm7861 predicted gene 7861 AY761184 cDNA sequence AY761184 122 10493831 S100a8 S100 calcium binding protein A8 (calgranulin A) 4.0382 Irgm2 immunity-related GTPase family M member 2 123 10376326 4.0222 Igtp interferon gamma induced GTPase 124 10492174 Tm4sf4 transmembrane 4 superfamily member 4 4.0029 125 10516064 Mfsd2a major facilitator superfamily domain containing 2A 3.9983 126 10495993 Elovl6 ELOVL family member 6, elongation of long chain fatty acids (yeast) 3.9754 Slc3a1 solute carrier family 3, member 1 127 10447264 3.9326 Prepl prolyl -like 128 10452770 Capn13 calpain 13 3.9264 129 10346668 Fam117b family with sequence similarity 117, member B 3.9235 130 10578857 Anxa10 annexin A10 3.8842 131 10542355 Emp1 epithelial membrane protein 1 3.7811 132 10544114 Hipk2 homeodomain interacting protein kinase 2 3.7730 133 10430372 Rac2 RAS-related C3 botulinum substrate 2 3.7719 134 10461439 Fads1 fatty acid desaturase 1 3.7640 135 10409278 Nfil3 nuclear factor, interleukin 3, regulated 3.7625 136 10467470 Aldh18a1 aldehyde dehydrogenase 18 family, member A1 3.7439 Gm7849 predicted gene 7849 Gm7861 predicted gene 7861 137 10577434 Gm14851 predicted gene 14851 3.7197 Defa-rs1 defensin, alpha, related sequence 1 AY761184 cDNA sequence AY761184 138 10498978 Lrat lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) 3.6999 139 10357488 Cd55 CD55 antigen 3.6812 140 10547153 Alox5 arachidonate 5-lipoxygenase 3.6810 141 10450800 Trim15 tripartite motif-containing 15 3.6748 142 10546962 Ghrl ghrelin 3.6727 143 10506433 Dab1 disabled homolog 1 (Drosophila) 3.6018 144 10517609 Cda cytidine deaminase 3.5674 145 10420988 Dpysl2 dihydropyrimidinase-like 2 3.5322 146 10585186 1600029D21Rik RIKEN cDNA 1600029D21 gene 3.5317 10351463 147 Rgs5 regulator of G-protein signaling 5 3.5231 10351455 148 10475653 Slc27a2 solute carrier family 27 (fatty acid transporter), member 2 3.5193 H2-T3 histocompatibility 2, T region locus 3 H2-T3-like MHC class I antigen 149 10450735 3.5162 H2-D1 histocompatibility 2, D region locus 1 H2-T18 histocompatibility 2, T region locus 18 150 10466008 Dak dihydroxyacetone kinase 2 homolog (yeast) 3.4970 151 10485405 Cd44 CD44 antigen 3.4922 152 10472097 Fmnl2 formin-like 2 3.4921 153 10477250 Hck hemopoietic cell kinase 3.4641 154 10525016 Tbx3 T-box 3 3.4567 155 10444312 Btnl2 butyrophilin-like 2 3.4509 156 10440513 Cyyr1 cysteine and tyrosine-rich protein 1 3.4321 157 10496023 Casp6 caspase 6 3.4140 158 10512904 Aldob aldolase B, fructose-bisphosphate 3.3880 159 10393573 Lgals3bp lectin, galactoside-binding, soluble, 3 binding protein 3.3847 160 10406176 Slc9a3 solute carrier family 9 (sodium/hydrogen exchanger), member 3 3.3712 161 10496638 Odf2l outer dense fiber of sperm tails 2-like 3.3320 162 10489204 Tgm2 transglutaminase 2, C polypeptide 3.3276 163 10466530 Pcsk5 /kexin type 5 3.3231 164 10366774 Avil advillin 3.3180 165 10485597 Depdc7 DEP domain containing 7 3.3066 Gm14851 predicted gene 14851 Defa-rs1 defensin, alpha, related sequence 1 166 10577421 AY761184 cDNA sequence AY761184 3.2753 Gm7849 predicted gene 7849 Gm7861 predicted gene 7861 167 10429555 2010109I03Rik RIKEN cDNA 2010109I03 gene 3.2696 168 10471880 Mir181b-2 microRNA 181b-2 3.2547 169 10466110 Ms4a10 membrane-spanning 4-domains, subfamily A, member 10 3.2492 170 10444244 Tap1 transporter 1, ATP-binding cassette, sub-family B (MDR/TAP) 3.2423 171 10472757 Cybrd1 cytochrome b reductase 1 3.2383 Socs2 suppressor of cytokine signaling 2 172 10394674 3.2008 Gm9847 suppressor of cytokine signaling 2 pseudogene 173 10399478 Lpin1 lipin 1 3.1918 174 10463282 Entpd7 ectonucleoside triphosphate diphosphohydrolase 7 3.1653 Ifi47 interferon gamma inducible protein 47 175 10375515 3.1548 Olfr56 olfactory receptor 56 176 10492136 Dclk1 doublecortin-like kinase 1 3.1517 177 10538214 D330028D13Rik RIKEN cDNA D330028D13 gene 3.1417 178 10456248 Onecut2 one cut domain, family member 2 3.1085 179 10459071 2010002N04Rik RIKEN cDNA 2010002N04 gene 3.0889 180 10585282 2010007H06Rik RIKEN cDNA 2010007H06 gene 3.0811 181 10390860 Krt23 keratin 23 3.0759 182 10523547 Agpat9 1-acylglycerol-3-phosphate O-acyltransferase 9 3.0759 Gsta1 glutathione S-transferase, alpha 1 (Ya) Gm10639 predicted gene 10639 183 10587331 3.0649 Gsta2 glutathione S-transferase, alpha 2 (Yc2) Gm3776 predicted gene 3776 Gm1502 predicted gene 1502 184 10545187 Gm8760 predicted gene 8760 3.0407 Igkv4-71 immunoglobulin kappa chain variable 4-71 185 10526514 Cldn15 claudin 15 3.0112 List of genes enriched in Colon No Affimetrix Transcripts Cluster ID Gene Symbol Gene Description Fold Change Graft/Colon 1 10497337 Car1 carbonic anhydrase 1  2 10476042 Tgm3 transglutaminase 3, E polypeptide  3 10463005 Cyp2c55 cytochrome P450, family 2, subfamily c, polypeptide 55  4 10568165 Zg16 zymogen granule protein 16  Saa1 serum amyloid A 1 5 10563611  Saa2 serum amyloid A 2 6 10424683 Ly6g lymphocyte antigen 6 complex, locus G  7 10519527 Abcb1a ATP-binding cassette, sub-family B (MDR/TAP), member 1A  8 10430873 Cyp2d34 cytochrome P450, family 2, subfamily d, polypeptide 34   9 10599008 Slc6a14 solute carrier family 6 (neurotransmitter transporter), member 14   10 10551531 Sycn syncollin   Selenbp1 selenium binding protein 1 11 10494114   Selenbp2 selenium binding protein 2 10503508 12 Ggh gamma-glutamyl   10503523 13 10500570 Hao2 hydroxyacid oxidase 2   14 10395201 Slc26a3 solute carrier family 26, member 3   15 10415446 Atp12a ATPase, H+/K+ transporting, nongastric, alpha polypeptide  16 10451016 Mep1a meprin 1 alpha   17 10547206 Fxyd4 FXYD domain-containing ion transport regulator 4  18 10411082 Thbs4 thrombospondin 4   19 10448402 Prss30 protease, serine, 30  20 10593198 Fam55b family with sequence similarity 55, member B   21 10568001 Sult1a1 family 1A, phenol-preferring, member 1   22 10478525 Wfdc2 WAP four-disulfide core domain 2  23 10500555 Hsd3b3 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta- 3  24 10592266 Slc37a2 solute carrier family 37 (glycerol-3-phosphate transporter), member 2  25 10557300 Aqp8 8  26 10514708 Insl5 insulin-like 5   27 10380614 Hoxb13 homeobox B13 

28 10531100 Sult1d1 sulfotransferase family 1D, member 1  29 10585194 Il18 interleukin 18  30 10391561 Pyy peptide YY  31 10469151 Itih5 inter-alpha (globulin) inhibitor H5  32 10471464 St6galnac6 ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 6  33 10365640 Slc5a8 solute carrier family 5 (iodide transporter), member 8   34 10407792 Gpr137b-ps -coupled receptor 137B, pseudogene  35 10377938 Eno3 enolase 3, beta muscle  36 10516484 Gjb3 protein, beta 3   37 10483074 Gcg glucagon   38 10354777 Satb2 special AT-rich sequence binding protein 2   Acsm3 acyl-CoA synthetase medium-chain family member 3 39 10556769   Eri2 2 40 10419034 2610528A11Rik RIKEN cDNA 2610528A11 gene  41 10459183 Slc26a2 solute carrier family 26 (), member 2   42 10514912 Dio1 deiodinase, iodothyronine, type I  43 10440647 Cldn8 claudin 8  44 10440655 2310079G19Rik RIKEN cDNA 2310079G19 gene  45 10494085 Selenbp2 selenium binding protein 2   46 10507137 Pdzk1ip1 PDZK1 interacting protein 1   47 10472946 Hoxd13 homeobox D13  48 10359422 Prdx6 peroxiredoxin 6  49 10574498 Ces2a 2A  50 10598771 Maoa monoamine oxidase A  51 10537690 Sval1 seminal vesicle antigen-like 1  52 10581824 Fa2h fatty acid 2-hydroxylase  53 10539156 Gm15401 predicted gene 15401  54 10575833 Hsd17b2 hydroxysteroid (17-beta) dehydrogenase 2  55 10538163 Abp1 amiloride binding protein 1 (amine oxidase, copper-containing)   56 10425772 Cyp2d12 cytochrome P450, family 2, subfamily d, polypeptide 12  57 10406407 Arrdc3 arrestin domain containing 3  58 10523451 Anxa3 annexin A3   59 10430319 Tst thiosulfate sulfurtransferase, mitochondrial   60 10394060 Sectm1b secreted and transmembrane 1B  61 10373600 Mettl7b methyltransferase like 7B   62 10467385 Cyp2c68 cytochrome P450, family 2, subfamily c, polypeptide 68   63 10376216 Slc36a1 solute carrier family 36 (proton/amino acid symporter), member 1   64 10523190 Parm1 prostate androgen-regulated mucin-like protein 1  65 10530145 Tlr1 toll-like receptor 1  66 10516490 Gjb5 , beta 5   67 10475990 Slc20a1 solute carrier family 20, member 1   68 10403291 Akr1c14 aldo-keto reductase family 1, member C14   69 10461934 Trpm6 transient receptor potential cation channel, subfamily M, member 6   70 10454192 Ttr transthyretin   71 10588263 Slco2a1 solute carrier organic anion transporter family, member 2a1  72 10366546 Cpm carboxypeptidase M  Cyp2c40 cytochrome P450, family 2, subfamily c, polypeptide 40 Cyp2c67 cytochrome P450, family 2, subfamily c, polypeptide 67 73 10467390 

Cyp2c68 cytochrome P450, family 2, subfamily c, polypeptide 68 Cyp2c69 cytochrome P450, family 2, subfamily c, polypeptide 69 74 10403312 Akr1c19 aldo-keto reductase family 1, member C19   75 10428796 Fbxo32 F-box protein 32   76 10466606 Anxa1 annexin A1   77 10367041 Rdh9 retinol dehydrogenase 9   10374453 78    Glul glutamate-ammonia (glutamine synthetase)   79 10574023 Mt2 metallothionein 2  80 10602756 Smpx small muscle protein, X-linked   81 10565958 P2ry6 pyrimidinergic receptor P2Y, G-protein coupled, 6   82 10536273 Casd1 CAS1 domain containing 1   83 10411373 Hexb hexosaminidase B   Nr1d1 nuclear receptor subfamily 1, group D, member 1 84 10390691  Thra thyroid hormone receptor alpha 85 10423333 Fam134b family with sequence similarity 134, member B  86 10598203 Ccl28 chemokine (C-C motif) ligand 28  87 10574780 Hsd11b2 hydroxysteroid 11-beta dehydrogenase 2  88 10515282 Tspan1 tetraspanin 1   89 10433445 Abat 4-aminobutyrate aminotransferase  90 10376376 1810065E05Rik RIKEN cDNA 1810065E05 gene  91 10574524 Ces2c carboxylesterase 2C  92 10514421 Lrrc19 leucine rich repeat containing 19   93 10590957 Fut4 fucosyltransferase 4  94 10349295 Tcfcp2l1 transcription factor CP2-like 1  95 10572949 Nr3c2 nuclear receptor subfamily 3, group C, member 2  96 10404059 Hist1h1c histone cluster 1, H1c  97 10580765 Pllp plasma membrane proteolipid   98 10437191 B3galt5 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 5   99 10469816 Il1rn interleukin 1 receptor antagonist   100 10391555 Ppy pancreatic polypeptide  101 10379176 Unc119 unc-119 homolog (C. elegans)  102 10496438 Adh1 alcohol dehydrogenase 1 (class I)  103 10490923 Car2 carbonic anhydrase 2  104 10354374 Slc40a1 solute carrier family 40 (iron-regulated transporter), member 1  105 10458704 Spink3 serine peptidase inhibitor, Kazal type 3  106 10429564 Ly6a lymphocyte antigen 6 complex, locus A  107 10479047 Pck1 phosphoenolpyruvate carboxykinase 1, cytosolic  108 10452202 Acer1 alkaline ceramidase 1  109 10580286 Best2 bestrophin 2  110 10390748 Tns4 tensin 4  111 10356968 Pam peptidylglycine alpha-amidating monooxygenase  Eif4ebp3 eukaryotic translation initiation factor 4E binding protein 3 112 10454944  Ankhd1 ankyrin repeat and KH domain containing 1 113 10492300 Aadac arylacetamide deacetylase ()  114 10501222 Gstm2 glutathione S-transferase, mu 2  115 10348062 B3gnt7 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 7   116 10422598 Sepp1 selenoprotein P, plasma, 1   117 10606868 Bex1 brain expressed gene 1  118 10495675 F3 factor III   119 10472958 Hoxd10 homeobox D10  120 10492021 Postn periostin, osteoblast specific factor  121 10432652 Cela1 -like family, member 1  122 10375667 Rnf130 ring finger protein 130  123 10501229 Gstm1 glutathione S-transferase, mu 1   124 10519886 Sema3c sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C   125 10589076 Slc25a20 solute carrier family 25 (mitochondrial carnitine/acylcarnitine ), member 20   126 10601484 2010106E10Rik RIKEN cDNA 2010106E10 gene   127 10529819 Fgfbp1 fibroblast growth factor binding protein 1   128 10431711 Slc2a13 solute carrier family 2 (facilitated glucose transporter), member 13   129 10511870 Fut9 fucosyltransferase 9   130 10466130 Ms4a8a membrane-spanning 4-domains, subfamily A, member 8A   131 10503259 Trp53inp1 transformation related protein 53 inducible nuclear protein 1  132 10388834 Slc13a2 solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 2   133 10514221 Plin2 perilipin 2   134 10501879 Usp53 ubiquitin specific peptidase 53   135 10445347 Clic5 chloride intracellular channel 5   136 10504743 Nans N-acetylneuraminic acid synthase (sialic acid synthase)   10348902 Gal3st2 galactose-3-O-sulfotransferase 2 137   10348896 Gm6086 predicted gene 6086 138 10422412 Slc15a1 solute carrier family 15 (oligopeptide transporter), member 1  139 10493794 S100a14 S100 calcium binding protein A14  140 10603746 Maob monoamine oxidase B  141 10497296 Slc10a5 solute carrier family 10 (sodium/bile acid cotransporter family), member 5   142 10411156 Scamp1 secretory carrier membrane protein 1   143 10365482 Timp3 tissue inhibitor of metalloproteinase 3   10362511 144 Gstm3 glutathione S-transferase, mu 3  10501218 145 10461856 Gna14 guanine nucleotide binding protein, alpha 14  146 10542872 Rps4y2 ribosomal protein S4, Y-linked 2  147 10599736 Fhl1 four and a half LIM domains 1   148 10430866 Cyp2d10 cytochrome P450, family 2, subfamily d, polypeptide 10   149 10548817 Plbd1 domain containing 1   150 10414065 Anxa8 annexin A8   151 10474809 Phgr1 proline/histidine/glycine-rich 1  152 10497149 Wls wntless homolog (Drosophila)  153 10541885 Scnn1a , nonvoltage-gated 1 alpha   154 10466521 Gcnt1 glucosaminyl (N-acetyl) transferase 1, core 2   Ces2d-ps carboxylesterase 2D, pseudogene 155 10574532 Ces2c carboxylesterase 2C   Ces2b carboxyesterase 2B 156 10357239 Tmem37 transmembrane protein 37   157 10436978 Cbr3 carbonyl reductase 3   158 10436945 Slc5a3 solute carrier family 5 (inositol transporters), member 3   159 10542822 Rep15 RAB15 effector protein  160 10482030 Stom stomatin  161 10421774 Dgkh diacylglycerol kinase, eta  162 10511975 Slc35a1 solute carrier family 35 (CMP-sialic acid transporter), member 1  163 10506658 Hspb11 heat shock B (small), member 11  164 10426891 Mettl7a1 methyltransferase like 7A1  Gm10035 predicted gene 10035 Mettl7a2 methyltransferase like 7A2 165 10426894   Mettl7a1 methyltransferase like 7A1 Higd1c HIG1 domain family, member 1C 166 10487011 Gatm glycine amidinotransferase (L-arginine:glycine amidinotransferase)  167 10538791 Tnip3 TNFAIP3 interacting protein 3   168 10437804 Pla2g10 phospholipase A2, group X  169 10544982 Nt5c3 5'-, cytosolic III  170 10439282 Csta cystatin A  171 10413086 Adk adenosine kinase  172 10411459 Tmem171 transmembrane protein 171  173 10434758 St6gal1 beta galactoside alpha 2,6 sialyltransferase 1  174 10469353 Fam23a family with sequence similarity 23, member A  175 10575328 Phlpp2 PH domain and leucine rich repeat   176 10461979 Aldh1a1 aldehyde dehydrogenase family 1, subfamily A1  177 10575184 Wwp2 WW domain containing E3 ubiquitin protein ligase 2   178 10494288 Golph3l golgi phosphoprotein 3-like  179 10365983 Lum lumican   180 10493789 S100a13 S100 calcium binding protein A13  181 10572928 Rasd2 RASD family, member 2  182 10402473 Clmn calmin  183 10556491 Far1 fatty acyl CoA reductase 1  184 10400143 Stxbp6 syntaxin binding protein 6 (amisyn)  185 10380599 Ttll6 tubulin tyrosine ligase-like family, member 6  186 10361828 Cited2 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2  187 10419578 Ndrg2 N-myc downstream regulated gene 2   188 10494761 Vtcn1 V-set domain containing T cell activation inhibitor 1   Tgoln1 trans-golgi network protein 189 10545450   Tgoln2 trans-golgi network protein 2 190 10544795 Mir196b microRNA 196b   191 10519607 4930420K17Rik RIKEN cDNA 4930420K17 gene   192 10365971 Btg1 B-cell translocation gene 1, anti-proliferative    193 10415572 2410022M11Rik RIKEN cDNA 2410022M11 gene   Mrps6 mitochondrial ribosomal protein S6 194 10436941   Slc5a3 solute carrier family 5 (inositol transporters), member 3 195 10389222 Ccl6 chemokine (C-C motif) ligand 6   Tesk2 testis-specific kinase 2 196 10507347   Toe1 target of EGR1, member 1 (nuclear) 197 10348906 Gm6086 predicted gene 6086   198 10467110 Lipo1 lipase, member O1   199 10537545 Gm7254 predicted gene 7254   200 10402347 Ifi27l2a interferon, alpha-inducible protein 27 like 2A   201 10410351 AA987161    202 10475532 Sqrdl sulfide quinone reductase-like (yeast)   203 10404359 Mboat1 membrane bound O-acyltransferase domain containing 1   204 10502732 Prkacb protein kinase, cAMP dependent, catalytic, beta    205 10434302 Klhl24     206 10481435 Ccbl1 cysteine conjugate-beta 1   207 10477169 Id1 inhibitor of DNA binding 1   Klk1 1 208 10552546 Klk1b5 kallikrein 1-related peptidase b5   Klk1b4 kallikrein 1-related pepidase b4 209 10480145 Rsu1 Ras suppressor protein 1