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SUPPLEMENTARY DATA “The receptor tyrosine kinase EPHB4 has tumor suppressor activities in intestinal tumorigenesis “ SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1: Immunostaining with endothelial markers. Immunohistochemical staining against CD34 (A-B, C-D) or CD105 (C-D) did not show significant differences between control HT29 cells transfected with pEGFP-N2 and derivative cells transfected with dominant negative EPHB4C-EGFP (A-D) or small intestinal tumors from Apcmin/+ mice that are wild type or heterozygous for EphB4 (E-F). Arrowheads indicate blood vessels. Supplementary Figure 2: Number and location of goblet, Paneth and enteroendocrine cells. Staining with 1% Alcian blue was used to reveal goblet cells. Paneth cells were detected by immunostaining with a rabbit polyclonal anti-lysozyme antibody (Dako, CA; prediluted) after treatment with 10 mM citrate pH 6 for 20 min for antigen retrieval. Enteroendocrine cells were quantified after Grimelius staining as described before (Fernandez Pascual et al 1976 Stain Technol 51:231). No differences were observed in the number and location of small intestinal goblet cells (Alcian blue positive; A-B), Paneth cells (Lysozyme positive; C-D) or enteroendocrine cells (Grimelius staining positive; E-F) of Apcmin/+;EphB4+/- and Apcmin/+;EphB4+/+ mice (n=10 and 7, respectively). Supplementary Figure 3: Microarray data validation. Histogram A) shows the gene expression differences observed in Apcmin/+;EphB4+/+ and Apcmin/+;EphB4+/+ mice using microarray analysis and quantitative Real-Time RT-PCR. B) An excellent correlation was observed between gene expression assessments made with these two techniques (Pearson ’s R=0.99 and p=0.003). Theprimers used were EhBEphB4-F: CAACTGGATGAGAGCGAGAG; EphB4-R: GAGGCAGAGAACTGCAATGA; Igfpbp5-F: AAGCTTCCCTCCAGGAGTTC; Igfpbp5-R: GGAGGGCTTACACTGCTTTC; Mmp2-F: GGGTGGTGGTCATAGCTACTT; Mmp2-R: TTCCAAACTTCACGCTCTTG; Col5a2-F; CAAGACACCTGCTCTAAGCG; Col5a2-R: CCAACATCTATGATGGGCAA; and for 18S rRNA the sequence of the primers and the internal probe were 18S-rRNA-F: AGTCCCTGCCCTTTGTACACA, 18S-rRNA-R: GATCCGAGGGCCTCACTAAAC and 18S-rRNA-probe: FAM-CGCCCGTCGCTACTACCGATTGG-TAMRA. Supplementary Figure 4: EPHB4 levels and Wnt signaling. A-D) Immunohistochemical staining with an anti-catenin did not reveal any significant differences in the levels or localization of -catenin. E) The levels of activity of TCF4/- catenin were assessed using the TOP/FOP system as previously reported (Korinek et al 1997 Science 275:1784). Briefly, cells were transfected with a vector containing a three tandem repeat of a consensus TCF4/-catenin binding site or a mutated binding site driving the transcription of a Luciferase reporter gene. The pRL-TK plasmid was used to control for transfection efficiency. No differences were observed in catenin/TCF4 trascriptional activity in empty vector control HT29 cells (EV) and EPHB4 dominant negative transfectants (DN). 1 SUPPLEMENTARY FIGURE LEGENDS (cont.) Supplementary Figure 5: Expression of genes involved in the adenoma to carcinoma transition. Previous studies have identified these eight genes as key players in the transition from adenoma to carcinoma (34). The fold change in the expression of these genes is shown for intestinal carcinomas/adenomas in Apcmin/+ mice (grey bars) and in adenomas from Apcmin/+ mice that are wild type or heterozygous for EphB4 (back bars). The inset shows the correlation between these values. Supplementary Figure 6: Functional network of 60 out of 183 genes differentially expressed in Apcmin/+ mice that are wild type or heterozygous for EPHB4. This network graph represents genes as nodes and functional connections as links. Genes down-regulated (red nodes), genes coding for collagen (blue nodes), genes coding for proteinases (green nodes) and genes coding for growth factors (orange nodes) are highlighted. The larger red node corresponds to EphB4. 2 SupplementaryFigure1 ABAntiCD34 AntiCD34 HT29 HT29 pEGFPN2 pEPHB4C EGFP C AntiCD105D AntiCD105 HT29 HT29 pEGFPN2 pEPHB4C EGFP EFAntiCD34 AntiCD34 Apcmin/+;EphB4+/+ Apcmin/+;EphB4+/ 3 SupplementaryFigure2 A) Alcian Blue B) Alcian Blue EphB4+/+;Apcmin/+ EphB4+/-;Apcmin/+ C) Anti-Lysozyme D) Anti-Lysozyme EphB4+/+;Apcmin/+ EphB4+/-;Apcmin/+ E) Grimelius F) Grimelius Apcmin/+;EphB4+/+ Apcmin/+;EphB4+/- 4 SupplementaryFigure3 A) 16 qPCR 12 Array nge 8 4 Fold cha Fold 0 MMP2 Col5a2 EPHB4 IGFBP5 B) Pearson’s R=0.99 p=0.003 5 SupplementaryFigure4 -catenin/TCF4 activity (Relative Luc levels) 0 2 4 6 HT29- EV HT29- DN E) 6 SupplementaryFigure5 Fold change -20 -15 -10 10 -5 0 5 S100g EphB4 het/wt Carcinoma/adenoma Sprr2f Sox17 Myl9 -10 Fold EphB4 WT/HET Mcpt2 -5 -20 -15 -10 -5 5 Pearson R:0.96 Igfbp5 Fold A/C 5 p<0.003 Serpine2 10 7 SupplementaryFigure6 COLOR KEY 8 EphB4inintestinaltumorigenesis Dopesoetal Supplementary Table 1: Genes with fold difference greater than 2-fold in adenomas of Apcmin/+ mice that are wild type or heterozygous for EphB4. Meanfoldin probeset Gene Symbol Gene Name EphB4WTvs. HET 1448964_at S100g S100calciumbindingproteinG 6,07 1448194_a_at H19 H19fetallivermRNA 5,91 1417600_at Slc15a2 solutecarrierfamily15(H+/peptidetransporter), 5,62 member2 1416040_at Lipf lipase,gastric 3,77 1453645_at 2700046A07Rik RIKENcDNA2700046A07gene 3,27 1449833_at Sprr2f smallprolinerichprotein2F 3,18 1419118_at 2900093B09Rik RIKENcDNA2900093B09gene 2,92 1459309_at Rufy3 RUNandFYVEdomaincontaining3 2,83 1445104_at E230029C05Rik RIKENcDNAE230029C05gene 2,82 1423935_x_at Krt114 keratincomplex1,acidic,gene14 2,82 1438519_at Klhl24 kelchlike24(Drosophila) 2,76 1449254_at Spp1 secretedphosphoprotein1 2,63 1439351_at A930031D07Rik RIKENcDNAA930031D07gene 2,53 1442489_at D1Ertd564e DNAsegment,Chr1,ERATODoi564,expressed 2,45 1446693_at RRR RRR 2,43 1436780_at Ogt OlinkedNacetylglucosamine(GlcNAc)transferase 2,42 (UDPNacetylglucosamine:polypeptideNR acetylglucosaminyltransferase) 1438676_at Mpa2l macrophageactivation2like///similarto 2,34 macrophageactivation2like///similarto macrophageactivation2like 1450165_at Slfn2 schlafen2 2,33 1452787_a_at Prmt1 proteinarginineNmethyltransferase1 2,31 1449286_at Ntng1 netrinG1 2,24 1449488_at Pitx1 pairedlikehomeodomaintranscriptionfactor1 2,18 1447845_s_at Vnn1 vanin1 2,06 1423954_at C3 complementcomponent3 2,07 1416619_at 4632428N05Rik RIKENcDNA4632428N05gene 2,09 1448748_at Plek pleckstrin 2,11 1441972_at 6230424C14Rik RIKENcDNA6230424C14gene 2,12 1416778_at Sdpr serumdeprivationresponse 2,14 1424733_at P2ry14 purinergicreceptorP2Y,Gproteincoupled,14 2,14 1416980_at Mettl7b methyltransferaselike7B 2,15 1424600_at Abp1 amiloridebindingprotein1(amineoxidase,copper 2,15 containing) 1418739_at Sgk2 serum/glucocorticoidregulatedkinase2 2,16 9 EphB4inintestinaltumorigenesis Dopesoetal (Continued) Meanfoldin probeset Gene Symbol Gene Name EphB4WTvs. HET 1435137_s_at 1200015M12Rik RIKENcDNA1200015M12gene///RIKENcDNA 2,27 1200016E24gene///RIKENcDNAA130040M12 gene///RIKENcDNAE430024C06gene 1430927_at Ceacam18 CEArelatedcelladhesionmolecule1 2,31 1425102_a_at Ace2 angiotensinIconvertingenzyme(peptidyl 2,31 dipeptidaseA)2 1423489_at Mmd monocytetomacrophagedifferentiationassociated 2,32 1416905_at Guca2a guanylatecyclaseactivator2a(guanylin) 2,34 1427054_s_at Abi3bp ABIgenefamily,member3(NESH)bindingprotein 2,34 1418237_s_at Col18a1 procollagen,typeXVIII,alpha1 2,34 1448741_at Slc3a1 solutecarrierfamily3,member1 2,36 1450852_s_at F2r coagulationfactorII(thrombin)receptor 2,37 1419498_at Tmigd transmembraneandimmunoglobulindomain 2,39 containing 1448823_at Cxcl12 chemokine(CXCmotif)ligand12 2,39 1416454_s_at Acta2 actin,alpha2,smoothmuscle,aorta 2,41 1427137_at Ces5 carboxylesterase5 2,42 1457721_at AI118064 ExpressedsequenceAI118064 2,43 1428909_at A130040M12Rik RIKENcDNAA130040M12gene 2,48 1455454_at Akr1c19 aldoketoreductasefamily1,memberC19 2,50 1426497_at Jarid1c jumonji,ATrichinteractivedomain1C(Rbp2like) 2,52 1446368_at 9130221J18Rik RIKENcDNA9130221J18gene 2,55 1423025_a_at Schip1 schwannomininteractingprotein1 2,55 1419703_at Col5a3 procollagen,typeV,alpha3 2,55 1426511_at Susd2 sushidomaincontaining2 2,56 1424245_at Ces2 carboxylesterase2///similartocarboxylesterase2 2,57 1421134_at Areg amphiregulin 2,59 1416194_at Cyp4b1 cytochromeP450,family4,subfamilyb,polypeptide 2,59 1 1449453_at Bst1 bonemarrowstromalcellantigen1 2,61 1451547_at 0610009A07Rik RIKENcDNA0610009A07gene 2,62 1448485_at Ggt1 gammaglutamyltransferase1 2,62 1451481_s_at Osta organicsolutetransporteralpha 2,63 1456733_x_at Serpinh1 serine(orcysteine)peptidaseinhibitor,cladeH, 2,64 member1 1418278_at Apoc3 apolipoproteinCIII 2,67 1456768_a_at RRR RRR 2,68 1418723_at Edg7 endothelialdifferentiation,lysophosphatidicacidG 2,68 proteincoupledreceptor7 1421489_a_at 2010106E10Rik RIKENcDNA2010106E10gene 2,69 10 EphB4inintestinaltumorigenesis Dopesoetal (Continued) Meanfoldin probeset Gene Symbol Gene Name EphB4WTvs. HET 1417976_at Ada adenosinedeaminase 2,70 1424768_at Cald1 caldesmon1 2,74 1424114_s_at Lamb11 lamininB1subunit1 2,75 1448755_at Col15a1 procollagen,typeXV 2,78 1443746_x_at Dmp1 dentinmatrixprotein1 2,79 1450641_at Vim vimentin 2,81 1422134_at Fosb FBJosteosarcomaoncogeneB 2,82 1452035_at Col4a1 procollagen,typeIV,alpha1 2,82 1424265_at Npl Nacetylneuraminatepyruvatelyase 2,84 1418601_at Aldh1a7 aldehydedehydrogenasefamily1,subfamilyA7 2,85 1437726_x_at C1qb complementcomponent1,qsubcomponent,beta 2,85 polypeptide 1449094_at Gja7 gapjunctionmembranechannelproteinalpha7 2,85 1417903_at Dfna5h deafness,autosomaldominant5homolog(human) 2,85 1448499_a_at Ephx2 epoxidehydrolase2,cytoplasmic
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    The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis

    Published OnlineFirst September 8, 2009; DOI: 10.1158/0008-5472.CAN-09-0706 Molecular Biology, Pathobiology, and Genetics The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis Higinio Dopeso,1,5 Silvia Mateo-Lozano,1 Rocco Mazzolini,1 Paulo Rodrigues,1 Laura Lagares-Tena,1 Julian Ceron,2 Jordi Romero,1,5 Marielle Esteves,1 Stefania Landolfi,4 Javier Herna´ndez-Losa,4 Julio Castan˜o,3 Andrew J. Wilson,6 Santiago Ramon y Cajal,4 John M. Mariadason,7 Simo Schwartz, Jr.,3,5 and Diego Arango1,5 Groups of 1Molecular Oncology, 2Functional Genomics and Genetics, and 3Drug Delivery and Targeting, Molecular Biology and Biochemistry Research Center (CIBBIM-Nanomedicine) and 4Department of Pathology, Vall d’Hebron Hospital; 5CIBER de Bioingenierı´a, Biomateriales y Nanomedicina, Barcelona, Spain; 6Department of Obstetrics and Gynecology, Vanderbilt University Medical Center, Nashville, Tennessee; and 7Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences, Austin Health, Heidelberg, Victoria, Australia Abstract Introduction Colorectal cancer is the second cause of cancer-related death Colorectal cancer is one of the leading causes of cancer-related in the western world, and although the genetic and molecular death in the western world and accounts for f1 million new mechanisms involved in the initiation and progression of these cases and f500,000 deaths every year worldwide. At the molecular tumors are among the best characterized, there are significant level, the constitutive activation of Wnt signaling is one of the gaps in our understanding of this disease. The role of EPHB hallmarks of colorectal cancer. Wnt activation most frequently signaling in colorectal cancer has only recently been realized.
  • Original Article Cytochrome P450 Family Proteins As Potential Biomarkers for Ovarian Granulosa Cell Damage in Mice with Premature Ovarian Failure

    Original Article Cytochrome P450 Family Proteins As Potential Biomarkers for Ovarian Granulosa Cell Damage in Mice with Premature Ovarian Failure

    Int J Clin Exp Pathol 2018;11(8):4236-4246 www.ijcep.com /ISSN:1936-2625/IJCEP0080020 Original Article Cytochrome P450 family proteins as potential biomarkers for ovarian granulosa cell damage in mice with premature ovarian failure Jiajia Lin1, Jiajia Zheng1, Hu Zhang1, Jiulin Chen1, Zhihua Yu1, Chuan Chen1, Ying Xiong3, Te Liu1,2 1Shanghai Geriatric Institute of Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China; 2Department of Pathology, Yale UniversitySchool of Medicine, New Haven, USA; 3Department of Gynaecology and Obestetrics, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China Received May 21, 2018; Accepted June 29, 2018; Epub August 1, 2018; Published August 15, 2018 Abstract: Premature ovarian failure (POF) is the pathological aging of ovarian tissue. We have previously established a cyclophosphamide-induced mouse POF model and found that cyclophosphamide caused significant damage and apoptosis of mouse ovarian granulosa cells (mOGCs). To systematically explore the molecular biologic evidence of cyclophosphamide-induced mOGC damage at the gene transcription level, RNA-Seqwas used to analyse the differ- ences in mOGC transcriptomes between POF and control (PBS) mice. The sequencing results showed that there were 18765 differential transcription genes between the two groups, of which 192 were significantly up-regulated (log2 [POF/PBS] > 2.0) and 116 were significantly down-regulated (log2 [POF/PBS] < -4.0). Kyoto Encyclopedia of Genes and Genomes analysis found that the neuroactive ligand-receptor interaction pathway was significantly up-regulated and metabolic pathways were significantly down-regulated in the POF group. Gene Ontology analy- sis showed that the expression of plasma membrane, regulation of transcription and ion binding functions were significantly up-regulated in the POF group, while the expression of cell and cell parts, catalytic activity and single- organism process functions were significantly down-regulated.
  • Genomic Signature of Parity in the Breast of Premenopausal Women

    Genomic Signature of Parity in the Breast of Premenopausal Women

    Santucci-Pereira et al. Breast Cancer Research (2019) 21:46 https://doi.org/10.1186/s13058-019-1128-x RESEARCH ARTICLE Open Access Genomic signature of parity in the breast of premenopausal women Julia Santucci-Pereira1*† , Anne Zeleniuch-Jacquotte2,3†, Yelena Afanasyeva2†, Hua Zhong2†, Michael Slifker4, Suraj Peri4, Eric A. Ross4, Ricardo López de Cicco1, Yubo Zhai1, Theresa Nguyen1, Fathima Sheriff1, Irma H. Russo1, Yanrong Su1, Alan A. Arslan2,5, Pal Bordas6,7, Per Lenner7, Janet Åhman6, Anna Stina Landström Eriksson6, Robert Johansson8, Göran Hallmans9, Paolo Toniolo5 and Jose Russo1 Abstract Background: Full-term pregnancy (FTP) at an early age confers long-term protection against breast cancer. Previously, we reported that a FTP imprints a specific gene expression profile in the breast of postmenopausal women. Herein, we evaluated gene expression changes induced by parity in the breast of premenopausal women. Methods: Gene expression profiling of normal breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers was performed using Affymetrix microarrays. In addition to a discovery/validation analysis, we conducted an analysis of gene expression differences in P vs. NP women as a function of time since last FTP. Finally, a laser capture microdissection substudy was performed to compare the gene expression profile in the whole breast biopsy with that in the epithelial and stromal tissues. Results: Discovery/validation analysis identified 43 differentially expressed genes in P vs. NP breast. Analysis of expression as a function of time since FTP revealed 286 differentially expressed genes (238 up- and 48 downregulated) comparing all P vs. all NP, and/or P women whose last FTP was less than 5 years before biopsy vs.