adipogenesis is inhibited by hyaluronidase.differentiation media (see methods) with or without the...
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![Page 1: Adipogenesis is inhibited by hyaluronidase.differentiation media (see methods) with or without the addition of 20 ug/ml of recombinant hyaluronidase (PEGPH20). (A) Cell numbers were](https://reader033.vdocuments.net/reader033/viewer/2022041813/5e591ea5a68d015bf933e100/html5/thumbnails/1.jpg)
Supplemental Figure 1.
Adipogenesis is inhibited by hyaluronidase. Mouse preadipocytes (3T3L1) were differentiated by the addition of adipocyte differentiation media (see methods) with or without the addition of 20 ug/ml of recombinant hyaluronidase (PEGPH20). (A) Cell numbers were analyzed by SRB assay and normalized to that of day 0 number. (B) Proliferative preadipocytes stained green with anti-BrdU. Scale Bar = 50 Microns. (C-E) Relative expression of mRNA for EBF1, Adipoq and Zfp521 at day 2 of differentiation. Data in A, C-E are represented usingbox-and-whiskers plots, with boxes representing the IQR, lines representing the median value, and whiskers representing minimum and maximum values, whereas data in D is represented as mean ± SEM; * P<0.05**, P < 0.01 (t test).
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Supplemental Figure 2.
Pref1 expression increases in colitis.
Representative histology stained with anti-PREF1/DLK of distal colon sections from control
mice or mice at day 0 and 7 days or 42 days after being provided with 3% DSS colitis in
drinking water. Brackets delineate submucosal region occupied by adipocytes. Scale Bar = 50
Microns.
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Supplemental Figure 3
Adipogenesis is coincident with HA accumulation in IBD patients.
Representative histology from colon sections from Crohn’s disease ((n=5). (A) Uninvolved
section from subject stained for Pref1. (B) Inflamed colon from the same patient stained for
Pref1. (C) Disease activity index score of IBD patients. Scale Bar = 20 Microns. Data in C is
represented usingbox-and-whiskers plots, with boxes representing the IQR, lines representing
the median value, and whiskers representing minimum and maximum values; ** P < 0.01,
*** P<0.001 (t test).
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Supplemental Figure 4
Hyaluronidase is effective to digest HA in skin and colon.
Representative histology of WT, Ella/hyal1 and PEGPH20 injected mice. (A, B) Normal
distal colon section of WT, Ella/hyal1 and PEGPH20 injected mice stained with (A)H&E and
(B) with HABP for HA. (C, D) Skin sections of WT, Ella/hyal1 and PEGPH20 injected mice
stained with (C) H&E and (D) HABP for HA. (E) HABP staining of colon at day 0 (control)
and 7 days after DSS administration and treated with PEGPH20
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Supplemental Figure 5
Hyaluronidase results in decreased macrophages and dendritic cells during DSS colitis.
(A to D) Flow cytometry analysis of colon lamina propria single cell suspensions showing
expression of (A, B) F4/80 and CD11B and (C, D) CD11C and MHC II from DSS d0
control, PEGPH20 d0, DSS d7 control and PEGPH20 treated DSS d7 mice. Numbers
represent the percentage of the cells in the indicated gate. Data in B and D are represented
usingbox-and-whiskers plots, with boxes representing the IQR, lines representing the median
value, and whiskers representing minimum and maximum values ; ** P < 0.01, *** P<0.001
(t test).
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Supplemental Figure 6
Incomplete inhibition of inflammatory response by PPARg agonist.
(A) Representative histology of distal colon sections in colitis of WT and BADGE treated
mice were stained with H&E. (B) Systemic bacteremia detected after colitis in mesenteric fat
and spleen from WT and with BADGE treatment (N=4 mice/group). (C) Endotoxin
concentration in the serum from WT and with BADGE treatment (N=4 mice/group). All error
bars indicate mean ± SEM; * P < 0.05, ** P < 0.01, *** P<0.001 (t test).
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Supplemental table 1 Up-‐regulated genes by DSS Down-‐regulated genes by DSSReg3b DbpSptssb CiartNos2 Lars2Plet1 Vaultrc5Apol10a Fut9Mmp3 Npy1rRetnlb Nxpe2Isx Reg4Lbp Cps1Clca4b Cttnbp2Cemip TrfSocs3 ChodlDhrs9 TefSlpi TtrTnf Snora23Mfsd2a JchainSlc3a1 Smoc2Aldh1a3 Slc16a12Nfil3 Gjb4Mmp13 Per3Tac1 Mir6236Chn2 Itih21810065E05Rik Plk3Ly6c1 Cdo1Cxcl5 Axin2Adra2a Snord22Acacb Tmprss13Wfdc18 Rnf32B3gnt6 Kyat1Slc5a9 Trpv6Rian Tppp3Serpina3n Igsf10Meg3 Pyroxd2Dnah17 2310079G19RikDuoxa2 Mptx2Duox1 Gsta3Car12 0610005C13RikB4galnt1 Per2Ttyh1 Fzd7Gm12250 CtgfTrim15 Tenm4Car8 Rn45sE2f2 HopxDuox2 Gjb5
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St3gal1 Slc2a5Mgat4c Timp4Agpat9 Atp10aSteap4 Fam46cPlat Sstr1Hmgcs2 Mptx1Ccl20 Sorcs2Cd177 Spin4Rnf150 Mir6516Nlrc5 Serpina9C2cd4b Apobec2Cmah Ces1fGda 2410006H16RikShank3 Prkar2bNupr1 Ace2Iigp1 Gstm1Mx1 CsadZbp1 Hist3h2aGm5431 TercGm14137 Car3Plaur 9230105E05RikCol23a1 Slc16a9Ptk6 Arl4dSyt6 Nr1d2Slc9a3 Hist1h1aDdah1 Eda2rGcg BambiSerpina3g Tmem132cLy6a PtproZc3h12a Pdzd7Cpn2 Hist2h2beGbp6 Scarna6Il1rl1 Hoga1Casc4 Gm14391Gpnmb Cldn2Snhg11 Glp2rIfit1 Cnn3Trim40 Ephb6Ifit2 Hist4h4Osmr Hist1h2bbLpo Ugt2b35Il18bp Aqp1Cdhr1 Celf4Muc2 HykkMuc1 Cox7a1
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Ffar4 5033404E19RikCldn8 Il33Casp4 Wee1Adamts1 Ces1eCeacam10 Scarna13Pla2g2a Dnah8Arhgap20 Col4a6Wtip Gm16576Npas2 Hist1h3bProx1 Dok7Rel Prdx4Apol6 Snora78Myrip Hist2h2bbBcl6 Rpph1Csf2rb Fam132aTdrd1 Csrnp1Cpne8 Hist1h2bm1700120E14Rik Sun2Ranbp17 CenppDmbt1 Epb41l4aosAqp4 Naaladl1Rsad2 PtpruIfi47 Snord17Slc7a9 Per1Cnn1 Amica1Nov Clec14aB3galt5 Hist1h2blFer1l4 Hist1h2bnAppl1 Hist1h1bPfkfb3 Hist1h3aCpne2 Crip1Igfbp5 Gstm6Tmem181c-‐ps Aldh1b1Cd38 Smim24Slc7a5 Paqr7Gm13363 Acer1Gm10400 Frem2Sec1 Etv4Zfhx2 Hist1h3eGbp5 Hist1h4hGbp4 Hist1h3cYpel2 Slc16a11Gpr22 Hist1h2bqRdh16 Hist1h2brC3 Hist1h2be
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Lrp8 Rgs10Ak4 Kyat3Des Hist1h3gC4b Phlda3Tnfrsf1b Fgfr31700024P16Rik Hist1h2adAdora1 Hist1h4cNcoa7 MyclF2rl2 Tmem14aTtn Snora74aSlc51b Gstm3Nckap5 Hist1h2apTmigd1 Hist1h2bkOasl2 Hist1h2bhSlc35d1 Itpripl2Nfasc Acyp1Bglap3 Efna4Cxcl13 Hist1h3dSlc13a2 Hist1h4fSlc13a2os Lipt2Pde9aHtr2bDnm3osSulf2Gna14Wfdc17Bhlha15Ceacam2EregCsf2rb2MgllMap3k8Fam196aMegf11Fut2Htra3Mapre3Cyp2c65Lgals9Clca3a1Fkbp5Sppl2aFcgr2bArmcx4Cp
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Pcsk5Trim72Ifit3bC77370Slc10a2LgmnD730005E14RikCol8a1Gm8615Il4raCeacam1Tnip3Tead1Socs1SynmBach2Arhgap27os3Gsdmcl-‐psItgamGm19461Tnfsf10Paqr8MgpRassf4Fam3bArhgap23XdhApol9aOgfrl1Tgtp2Ccl9Mmp28Actg2Kcnn3Stc2Nlrp9bGnpda1Prps1Hspa1aTaglnHmox1Galnt18Piezo1PcloSidt1
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GemLypd6bNt5c1aHcn2Cwh43Ttc7bKctd12Hspa1b4931406H21RikLy6c2AI661453Mbd6Itga2Zdhhc13Slc4a7Pgap1Pla2g5Ang44930565N06RikMuc4Ppm1lSgk2Mospd3Hcls1Il18Isg15Arap3Tmem139Tmem38bEmp1Auts2Kcnk6Cd68Pdlim3Glipr2Vcam1Pde8aRelnCry1Slc30a10Muc13Sod3Plekhm1Tbc1d9Pstpip2
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Tgtp1ArntlPrr139930111J21Rik2Tnfaip2Tc2nSlc2a4Tpm2Grin1osMboat2Ermp1Ido1EcscrSatb1Cdx2Pag1Oas3Gramd32010016I18RikNol4lFzd3Nkain1Slc36a4Slc5a3Insl3Fam219aBrd4Gm13889Igsf6Themis2D17H6S56E-‐5NfkbizNlgn2Evi2bTanc2Stat2Setd1bRrbp1Rbms3TifaApobCnnm2PappaFybFgl2
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Abca5Gnao1Slc28a3Tnfsf13bCyp2d9Pcdhgb2Ffar2Lrrc32Hivep3IgtpAdrbk2PdpnGalntl6Plxnc1AregNeat1Lonrf3Flt4Prdm1Ier5EtnpplRdh18-‐psTmcc3Leo1Icam1Ttyh3Lrrtm2Dclk1DaglbKmt2dSyvn1Slc8a1A330023F24RikEid3Ifit3AW112010Atn12210407C18RikMafbLhfpl2Pgs1Ehd4NbeaCyp2d10Dmxl2
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H2-‐T24ChadRobo2Grem1Sema7aHyou1TifabCfhArid3aCd84Ttc22Gprc5aBasp1HckLaptm5Adap2Adcy2Parp14UbdPolqStrip2Pmepa1Cntnap1RbpmsPgm5Ripk3Cd472610528A11RikCremArhgap35Alox5Camk2n14732471J01RikMacf1Galnt3Ralgapa2Ccdc97Zfp9St8sia5Cacna1bIrgm2Spon1Slit2Gbp2Gm15708
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Ano7Tgm2B3gnt5Zfand5Slc9a1Pcp4l1Ppp2r1bBmp7Anks6Bcl2l11Srgap1Abcc5Itgb2lSrrm2Ptprn2Tmc5Atp8a19130008F23RikAbcg8KaznMyo1dAbhd6Eif4g3Proser1Rims2Hap1Pom121Fam124aEfnb2PpyTtll10Nus1Fndc3bAcadlExoc6bNos1AW551984Scarf2Alg3Tspan6Plod2Fam122a6430548M08RikPtafrScg3
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FbrsMpzl1Dtx3lJak3Ryr2FryCacna1hTlr4Igf2bp2Ifit1bl1Arl14Rdh19Mroh3Pnrc1Tmem184cIfit1bl2Pdlim7Arhgap17Cmtm8Eif4e3Psmb8Tbrg3Dock4Abi2Tinagl1Mvb12bRdh9Srrm1Hk2Clec7aUnc5aRipk2C1galt1Iqgap2Rtp4RhojSlco3a1Tmem220Pcsk1nPcdhgb7Prr12PpifosRnf44Aoc2Srgap3
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Pglyrp1Hipk2Ssh2Kdm7aRnf39Pappa2HgfSema4aSrcapCelsr3Bcl3Krt84Gdap104930594C11RikSh3kbp11190002N15RikTrafd1Atp10bKlhdc8aFabp2Tmem131StambpKmt2aDvl3Trim12aMmp15Mpp3Kirrel3SpenB230354K17RikPlagl2B4galnt2A430088P11RikHspa5Acer2St53110062M04RikNcor2Asap1Grb10TprnPlxna4ItgavNeto2Ankrd22
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Lpin2Osbpl7Phactr2
10-‐SepTrim36Cyp2c55Tnfrsf11aApol7aZdhhc95830444B04Rik1110059E24RikSema6bPcdhga9Syn3Capn13Tex9Fth1Cd14Cpeb2Dcp2Slc16a6Creb3l1Ugt1a6bGabbr1Zfp711Trp53i11Tlr2Ubqln2Rimbp2Apaf1Lgals3bpCol18a13110043O21RikPtpn23Tbc1d15Fgd5PtprnHip12010109I03RikMs4a8aPtp4a1Prom1Hkdc1Rab6bFxyd6
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Nr1h4GalcCnbd2Creld2PyyFignl2Fscn1B130006D01RikAbhd17bNectin1Irf7FasIckGanRsc1a1Asap2CapgSec24aAnkrd28Tcf23Prrc2a5330417C22Rik
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Supplemental table 2 Up-‐regulated genes by PEGPH20 Down-‐regulated genes by PEGPH20Lars2 Mmp13Mir6236 Tmigd1Cxcl5 SptssbRn45s CemipPlat Reg3bHspa1a Gm13363Hspa1b B3gnt6Trf Mmp3Syt8 Creb3l3Chodl Cyp2c65Cttnbp2 1810065E05RikTmprss13 Ptgs2Reg4 LpoSlc16a12 Slc3a1Pyroxd2 Mt2Fam46c Ido1Ankrd37 Ifit2Nxpe2 RetnlbFut9 CmahMptx2 Il1rl1Steap4 RianHoxd10 Car8Banp Slc7a9Snord22 GcgGatm Cd177Smoc2 GchfrMptx1 Ifit1Tmem173 Adap2Slc4a4 Serpina3nDbp Plet1Fzd7 EregCiart Cyp2d10
Iigp1Emp1SlpiC4bApol9aLy6c1Ccl20Ccr2St3gal1Zfp37MgllNcoa7
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Adra2aCyp2d11Hmgcs2PpyGm5431PappaChn2Aldh1a3Col18a1Ccr1Mboat2Gadd45aTcf23PdpnCpAcacbMs4a6cKrt23Cyp2d9Igfbp5Isg15Adamts1Zg16GpnmbRac2B4galnt1Spink1TnfMyripCyp4f14Casc41500009L16RikAldh3b1Entpd1Apold1Bmp7Nt5c1aFabp2ItgamRsad2Apol10aApol6Gm14137EdnrbDpep1
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Fcgr3C3Ces2bMapre3Hsd3b2Mmp19Rab37Mep1bTex9Sema3aIfi47C77370Ifit3Slc2a4Ccl9Fkbp5Ppfia3Bglap3Aqp8Svep128-‐FebHsd3b3Pde4bGngt2Srxn1Ttyh1DcnMgat4cTekPtproCdh13Clec7aFbxo44Ranbp17Serpina3gNat8f2ItgalAspaTmem37Apobec11700052K11RikPpp3ccPla2g15Nat8f4Neto2
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9930111J21Rik2EsamGm12250Slfn5Ccdc122Hbb-‐b2Hbb-‐btTnfaip2Gbp4Ces2cGbp6Apoc2Dnase2aUgt1a1Lama4Car1Ube2l6Hba-‐a2Clca4bAntxr1Prss30Ccrl2Ceacam10Ces2d-‐psCes2eLyve1Prex1Chst11Rgs13PlekDgat2Hbb-‐b1Hbb-‐bsIfit3bGgt12210407C18RikAW112010LeprUbdSt8sia4Gm20939IsxIgfbp7Lgals9Prom1
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Pdlim2H2-‐Q1CfhRgl1CapgMt1Loxl2Dhx58EspnSgk1BikCasp3Zfp820Alox5apCsf2rbCar4Sod3TyrobpAgpat9Arl14Tstd3Vav3GptSpata6Mmrn1SrgnTff3Spns2Gm3336Btnl6Adgrl3Ppm1mTgm2Kremen2Pld4Trim6MgpSlc16a6Sepp1P3h4Abcg2Nrp1Ly6aCd84Ang4
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Dusp10Cxcl12Alox5Slc26a3Cd53Gm5141Rtp4Kctd18Chic1Slc25a34GlrxA630066F11RikSlc30a1Col12a1Cyp2c55C1qtnf1C1galt1c1Gpcpd1Sectm1bOgfrl1Saa2Sult1d1Abhd6Zfp52Glipr2Hkdc1Lypd8Ly6c2SlaGghC2Meg3Pcsk5Lrrc19Zfp781Clec2eVcam1Bcap29Herc6AI507597Sult1b1PostnItga8Pax8Psmb8
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Lyz2RbksGbp2Slc34a2Nceh1ArntlIgfbp4Psmb9BC021614Rasgrf2Itga4GemTspan6Il10raCwh43Plekho2Rbms3Zfp994Hip1Sema7aCar12DcxrHmox1C2cd4bPla2g3Hsdl2Lpin2Tnfsf13bZfp995AfmidSdhaf1Gdpd2Ifi27l2aH2-‐DMaCidebCd93IcoslDock4Pgap14931406C07RikMmp2Mrc1St3gal3Scarf2Snx20
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