Transcriptional Regulation of Pathways Activated by Lipopolysaccharide in RAW 264.7 CellsSangdun Choi*, Yun Anna Cao*, Sun Young Lee*, Mi Sook Chang*, Xiaocui Zhu*, Meghdad Rahdar†, Robert Hsueh†, Paul Sternweis†, and Melvin Simon*

*Molecular Biology Laboratory, Alliance for Cellular Signaling, Division of Biology, California Institute of Technology, Pasadena, California †Cell Preparation and Analysis Laboratory, Alliance for Cellular Signaling, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas

(interferon regulatory factor-3)

TIR (Toll/interleukin-1 receptor) domain

(TANK-binding kinase-1)

TIR domain-containing adapter-inducing IFN-beta

2004, Semi in Immunol 16:3

MyD88 dependent pathway

MyD88 independent pathway

triacylated lipopeptidediacylated lipopeptidetriacylated lipopeptide

diacylated lipopeptide

LPSResiquimod (R-848)Imidazoquinoline

dsRNA

CpG DNA

TLR (Toll-like receptor)-mediated Signaling Pathways

Regulatory Elements analyzed by Ingenuity Pathway Analysis

**

Regulatory Elements Gene Time CopyNFkB IRF3 IL1 1hr 2hr 4hr 8hr 16hr 48hr /Cell

Egr1 Egr1 Egr1 Egr1 _ Early growth response 1 (M22326)(1590) 5.13 1.49 2.25 1.98 1.06 2 29Scyb2 Scyb2 Scyb2 Scyb2 _ Small inducible cytokine subfamily, member 2 (X53798)(3347)_cxcl2 4.96 4.95 4.82 4.2 4.39 3.09 112MAIL MAIL MAIL MAIL _ Mus musculus mRNA for MAIL, complete cds (AB020974)(18515) 4.57 3.61 2.48 2.73 2.33 1.36 24Irg1 Irg1 Irg1 Irg1 _ Immunoresponsive gene 1 (L38281)(190) 4.19 5.09 5.78 5.49 4.57 3.65 88Egr2 Egr2 Egr2 Egr2 _ Early growth response 2 (X06746)(1574) 3.79 1.42 0.55 1.11 1.02 0.98 9CD83 CD83 CD83 CD83 _ Mus musculus CD83 antigen (Cd83), mRNA (NM_009856)(10633) 3.61 3.79 1.89 1.32 1.41 0.41 80Dusp2 Dusp2 Dusp2 Dusp2 _ Mus musculus dual specificity phosphatase 2 (Dusp2), mRNA (NM_010090)(1391)3.54 2.36 1.53 2.48 2.24 0.85 82Tdag Tdag Tdag Tdag _ T-cell death associated gene (U44088)(4702)_DT1P1B11, PHLDA1, TDAG51 3.37 3.04 1.95 2.47 2.35 1.57 17Scyb10 Scyb10 Scyb10 Scyb10 _ Small inducible cytokine B subfamily (Cys-X-Cys), member 10 (M86829)(106)_cxcl10, IP103.16 3.86 3.74 3.04 3.61 2.35 170Btg2 Btg2 Btg2 Btg2 _ B-cell translocation gene 2, anti-proliferative (M64292)(15706) 3.04 1.04 0.53 0.83 -0.6 -0 226Ifnb Ifnb Ifnb Ifnb _ Interferon beta, fibroblast (NM_010510)(12557) 3 3.58 3.94 4.3 3.07 0.59 9Lynx1 Lynx1 Lynx1 Lynx1 _ Mus musculus Ly6/neurotoxin 1 (Lynx1), mRNA (NM_011838)(6065)_MGC40364, SLURP22.97 2.93 3.5 3.81 3.57 2.55 25IL1b Il1b Il1b Il1b _ Interleukin 1 beta (M15131)(3465) 2.94 4.36 4.62 4.04 5.05 3.79 39Ifit3 Ifit3 Ifit3 Ifit3 _ Mus musculus interferon-induced protein with tetratricopeptide repeats 3 (Ifit3), mRNA (NM_010501)(2881)_Garg49, IFIT42.9 2.49 2.98 3.37 2.81 2.31 6Planh1 Planh1 Planh1 Planh1 _ Plasminogen activator inhibitor, type I (M33960)(15739)_SERPINE1 2.89 2.34 1.56 2.3 3.11 3.53 183Myc Myc Myc Myc _ Myelocytomatosis oncogene (X01023)(6265)_C-MYC 2.79 3.42 3 3.66 3.47 2.47 19Tnf Tnf Tnf Tnf _ Tumor necrosis factor (X02611)(15709)_TNF ALPHA, TNFSF2, Tnfsf1a 2.74 2.33 2.26 2.11 1.82 1 149Traf1 Traf1 Traf1 Traf1 _ Mus musculus Tnf receptor-associated factor 1 (Traf1), mRNA (NM_009421)(10571)2.73 2.73 1.64 1.55 2.13 1.93 30Csf3 Csf3 Csf3 Csf3 _ Colony stimulating factor 3 (granulocyte) (NM_009971)(6317) 2.66 2.91 3.4 3.49 4.41 2.7 12Pim1 Pim1 Pim1 Pim1 _ Mus musculus proviral integration site 1 (Pim1), mRNA (NM_008842)(13775)_Pim2.64 3.18 2.73 2.37 2.33 0.76 62Sei1 Sei1 Sei1 Sei1 _ P34SEI-1 (AF218291)(16089)_CDK4-binding protein p34SEI1 2.47 1.7 0.9 1.3 -0.8 0.79 110Scya4 Scya4 Scya4 Scya4 _ small inducible cytokine A4 (AF128219)(13530)_CCL4, MIP-1-BETA, ACT2, MIP-1B2.43 3.37 3.5 3.79 2.76 1.73 306Gpr84 Gpr84 Gpr84 Gpr84 _ Orphan G protein-coupled receptor 84 (NM_030720)(18189) 2.35 2.6 1.95 1.04 0.75 -0.2 366Tnfrsf5 Tnfrsf5 Tnfrsf5 Tnfrsf5 _ Tumor necrosis factor receptor superfamily, member 5 (M83312)(9525)_CD40, CD40L RECEPTOR, CD40L, TRAP, p502.19 2.57 3.48 3.76 2.11 1.62 14Zfp36 Zfp36 Zfp36 Zfp36 _ Zinc finger protein 36 (M58691)(15710)_ZINC FINGER TRANSCRIPTIONAL REGULATOR2.14 0.85 1.84 2.05 2.81 2.14 155Arc Arc Arc Arc _ Activity regulated cytoskeletal-associated protein (AF162777)(3568)_Arc3.1, KIAA0278, rg3.12.07 0.68 0.01 0.48 -0.2 0.44 21Rad Rad Rad Rad _ Ras-like GTP-binding protein rad (AF084466)(493)_RAD1, REM3, RRAD 2.03 2.77 1.09 0.56 0.67 0.51 74Ap2 Ap2 Ap2 Ap2 _ Adipocyte protein aP2 (K02109)(7874)_FABP4, A-FABP, A-fab, ALBP/Ap2, Albp, Ap4, Lbpl2.02 2.13 2.2 2.65 2.87 3.11 36Ier2 Ier2 Ier2 Ier2 _ Immediate early response 2 (M59821)(7933)_ETR101, ETR11, Growth factor-inducible protein, ch1, pip921.99 0.57 0.54 0.6 0.67 0.11 114Pde4b Pde4b Pde4b Pde4b _ Phosphodiesterase 4B, cAMP specific (AF208023)(1774) 1.98 2.3 1.43 0.61 1.47 0.86 12Clecsf9 Clecsf9 Clecsf9 Clecsf9 _ C-type (calcium dependent, carbohydrate recognition domain) lectin, superfamily member 9 (AB024717)(11367)_MINCLE1.96 2.04 1.94 1.41 1.71 0.93 130Tnfsf9 Tnfsf9 Tnfsf9 Tnfsf9 _ Tumor necrosis factor (ligand) superfamily, member 9 (L15435)(5106) 1.95 1.32 0.61 0.66 1.27 0.22 343Cash Cash Cash Cash _ Caspase homolog (Y14041)(7907)_C-FLIP, CFLAR, CLARP, Casper, FLAME, FLAME-1, FLIP, I-FLICE, MRIT, USURPIN1.95 1.49 1.47 1.51 1.85 0.98 121Ptpn16 Ptpn16 Ptpn16 Ptpn16 _ Mus musculus protein tyrosine phosphatase, non-receptor type 16 (Ptpn16), mRNA (NM_013642)(10684)_DUAL-SPECIFICITY PHOSPHATASE 1, DUSP1, Growth factor-inducible immediate early, HVH1, MKP-1, PTP Non-Receptor Type16 , PTPN10, Ptpn16, erp1.92 0.82 -0.3 0.89 1.12 1.52 34Gp49b Gp49b Gp49b Gp49b _ Glycoprotein 49 B (U05265)(9726)_CD85F, ILT11, LILRB7, LIR9, gp49 1.89 2 2.51 2.92 3.48 1.95 478Gp49a Gp49a Gp49a Gp49a _ Glycoprotein 49 A (M65027)(5152)_Gp49 1.89 2.04 2.56 3.08 3.71 2.11 432C3 C3 C3 C3 _ Complement component 3 (K02782)(6549)_ASP, Plp 1.87 2.11 2.19 2.14 1.29 0.75 36IL10 IL10 IL10 IL10 _ Mus musculus interleukin 10 (Il10), mRNA (NM_010548)(4474) 1.87 2.14 1.28 1.76 1.4 1.96 8Mlp Mlp Mlp Mlp _ MARCKS-like protein (X61399)(17309)_CSRP3, MARCKS RELATED PROTEIN, MMLP, Mrp1.78 1.88 2.15 1.94 1.25 0.81 496Ier3 Ier3 Ier3 Ier3 _ Immediate early response 3 (X67644)(12928)_DIF-2, GLY96, IEX-1 1.78 1.68 1.51 1.51 0.14 0.5 1018Maff Maff Maff Maff _ V-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) (AB009694)(3688)1.75 0.92 0.34 0.39 0.1 0.4 8Siah2 Siah2 Siah2 Siah2 _ Mus musculus seven in absentia 2 (Siah2), mRNA (NM_009174)(2825) 1.75 1.02 0.49 0.89 -0.4 0.47 23Tnfaip3 Tnfaip3 Tnfaip3 Tnfaip3 _ Mus musculus tumor necrosis factor, alpha-induced protein 3 (Tnfaip3), mRNA (NM_009397)(2887)1.73 2.01 1.71 1.02 1.28 0.61 33Saa3 Saa3 Saa3 Saa3 _ Serum amyloid A 3 (NM_011315)(14790) 1.72 2.83 4.01 4.16 4.71 3.13 74

2. Early Responses (1hr)

Gene Time CopySymbol Name 1hr 2hr 4hr 8hr 16hr 48hr /Cell

Ifi202a Ifi202a _ Interferon activated gene 202A (M31418)(12729) 0.1 0.6 4.7 6.2 5.8 5.1 27Ifit2 Ifit2 _ Interferon-induced protein with tetratricopeptide repeats 2 (U43085)(14057) -0.3 0.4 4.3 4.7 4.3 3.3 104Gbp3 Gbp3 _ Guanylate nucleotide binding protein 3 (U44731)(4230) 0.3 0.9 4.2 5.1 4.6 4.7 23Gbp1 Gbp1 _ Guanylate nucleotide binding protein 1 (M63961)(75) 0.1 0.6 4 4.7 3 3.2 18Ifi204 Ifi204 _ Interferon activated gene 204 (M31419)(12878) 0.3 0.7 3.6 4.7 4.1 3.6 53Tgtp Tgtp _ Mus musculus T-cell specific GTPase (Tgtp), mRNA (NM_011579)(10743) -0.1 0.2 3.5 4.8 2.7 3.3 9Usp18 Usp18 _ Ubiquitin specific protease 18 (NM_011909)(3505) -0.1 0.4 3.4 3.8 2.7 2.6 300Ifi1 Ifi1 _ Interferon inducible protein 1 (U19119)(3539) -0.2 0.9 3.2 3.2 2 2.3 384Mx1 Mx1 _ Myxovirus (influenza virus) resistance 1 (M21038)(6573) -0 0.4 3 4.1 3.4 3.6 11Gbp2 Gbp2 _ Guanylate nucleotide binding protein 2 (AF077007)(8212) 0.2 0.3 3 3.8 2.2 2.3 12Igtp Igtp _ Interferon gamma induced GTPase (U53219)(12523) -0.1 0.8 3 3.3 1.8 2 81Procr Procr _ protein C receptor, endothelial (AK007221)(787) 0.1 0.1 2.5 3.3 2.2 2.3 81Abcb2 Abcb2 _ ATP-binding cassette, sub-family B (MDR/TAP), member 2 (NM_013683)(5402) -0.1 0.1 2.4 2.9 1.9 1.8 201Slfn4 Slfn4 _ Schlafen 4 (AF099976)(16153) 0.1 0.4 2.4 3.3 4.1 4.8 69Stat2 Stat2 _ Signal transducer and activator of transcription 2 (AF206162)(14359) -0.5 -0 2.3 2.7 1.6 1.6 79Uty Uty _ ubiquitously transcribed tetratricopeptide repeat gene, Y chromosome (AK003586)(17850)0 0.3 2.3 2.5 1.5 1.4 504Dlm1 Dlm1 _ Tumor stroma and activated macrophage protein DLM-1 (AF136520)(545) 0.1 0.3 2.3 3.6 3.7 3 21Stat1 Stat1 _ Signal transducer and activator of transcription 1 (U06924)(4979) -0.1 0.2 2.2 3 2.3 2.3 304Rpt1 Rpt1 _ Regulatory protein, T lymphocyte 1 (J03776)(12027) -0.1 0.1 2.2 3 3 3.3 12Daxx Daxx _ Fas death domain-associated protein (AF006040)(11242) 0 0.5 2.1 2.4 1.4 1.3 148Ifi203 Ifi203 _ Interferon activated gene 203 (AF022371)(4990) -0.1 -0 2.1 3 2.1 2.1 15Elp4 Elp4 _ Mus musculus toll-like receptor 3 (Tlr3) mRNA, complete cds (AF355152)(8649) -0 -0 2.1 3.2 1.7 1.8 16Trex1 Trex1 _ Three prime repair exonuclease 1 (AF151106)(14346) 0.1 0.7 2.1 2.4 1.9 1.6 95Iigp Iigp-pending _ Interferon-inducible GTPase (AF194871)(330) 0 0.1 2 3.4 1.7 2.2 11Lgals9 Lgals9 _ Lectin, galactose binding, soluble 9 (U55060)(8078) 0.1 0.2 2 3.1 2.7 2.3 53Cish1 Cish1 _ Cytokine inducible SH2-containing protein 1 (NM_009896)(42) 0.1 0.8 2 1.7 1.1 1 59Irf7 Irf7 _ Interferon regulatory factor 7 (U73037)(14096) 0 0.4 2 2.7 2.5 2.8 213Oasl Oasl _ 2'-5' oligoadenylate synthetase-like (AF068835)(8249) -0.1 0.1 2 2.9 2.8 2.7 183Ldh3 Ldh3 _ lactate dehydrogenase 3, C chain, sperm specific (AK015214)(4208) 0.1 0.1 1.9 2.8 1.9 1.6 6Rsdr1 Rsdr1-pending _ Retinal short-chain dehydrogenase/reductase 1 (X95281)(9600) -0.1 0.8 1.8 2.2 3 1.2 38Lama4 Lama4 _ laminin, alpha 4 (NM_029509)(11555) 0.2 -0 1.8 3.1 2.2 2.3 9Gsdm Gsdm _ gasdermin (AK009862)(841) -0 0.2 1.8 2 0.8 0.9 326Mmp19 Mmp19 _ matrix metalloproteinase 19 (NM_025681)(17787) 0.1 0.5 1.7 2.1 3.9 1.6 18Axl Axl _ AXL receptor tyrosine kinase (X63535)(4800) 0 0.7 1.7 2.5 3 2.7 18Ci1 cAMP inducible 1 protein (Ci1) mRNA, complete cds (AF121080)(16014) 0.4 0.9 1.7 2.2 2.9 2.4 153Il13ra2 Il13ra2 _ Interleukin 13 receptor, alpha 2 (U65747)(9675) 0.4 0.8 1.7 2.4 1.7 0.4 9UbcM8 UbcM8 _ E2 ubiquitin conjugating enzyme (AF159230)(6603) -0 0.2 1.7 2.7 1.8 1.7 170Ifi47 Ifi47 _ Interferon gamma inducible protein, 47 kDa (M63630)(12833) -0.2 -0.4 1.7 2.6 0.7 1.5 153Scyb11 Scyb11 _ Small inducible cytokine subfamily B (Cys-X-Cys), member 11 (AF179872)(9970)0.2 0.7 1.7 1.6 2.8 1.7 21Sp100 Sp100 _ Nuclear antigen Sp100 (U83630)(6503) -0.1 -0.1 1.7 2.8 2 1.8 80Eif2ak2 Eif2ak2 _ Eukaryotic translation initiation factor 2 alpha kinase 2 (NM_011163)(12614) -0.1 0.1 1.6 2.1 1.2 1.3 282Katnb1 Katnb1 _ katanin p80 (WD40-containing) subunit B 1 (AK019472)(11631) 0.3 0.8 1.6 1.5 1.4 0.7 133Gch Gch _ GTP cyclohydrolase 1 (L09737)(16737) 0.2 0.7 1.6 2.4 1.6 1.2 227Fcgr2b Fcgr2b _ Fc receptor, IgG, low affinity IIb (NM_010187)(13688) 0.1 0.6 1.6 2 3 1.5 42

3. Intermediate Responses (4hr)

Gene Time CopySymbol Name 1hr 2hr 4hr 8hr 16hr 48hr /Cell

Lcn2 Lcn2 _ Lipocalin 2 (X81627)(12748) 0.4 0.1 0.1 0.6 3.6 3.4 7Gstt1 Gstt1 _ Glutathione S-transferase, theta 1 (X98055)(1709) 0.1 0.2 0.2 0.6 3.6 3.6 26Sqrdl Sqrdl _ Sulfide quinone reductase-like (yeast) (NM_021507)(15134) 0.2 -0 0.1 0.4 3.1 1.3 14Gp38 Gp38 _ Glycoprotein 38 (M73748)(9477) 0.1 0.1 0.1 0.8 2.9 3.3 9Aqp9 Aqp9 _ Aquaporin 9 (NM_022026)(11586) -0.1 0.5 0.9 0.8 2.9 1.8 15Macs Macs _ Myristoylated alanine rich protein kinase C substrate (M60474)(12809) 0 0.5 0.9 0.9 2.9 0.8 6Fbp LOC59010 _ Flavo-binding protein (AF174535)(9781) -0 -0 0.1 0.5 2.7 1.2 14Gdf15 Gdf15 _ Growth differentiation factor 15 (NM_011819)(11311) 0.3 0.4 0.1 0.2 2.7 2.1 19Procr Procr _ Protein C receptor, endothelial (L39017)(4797) 0.1 0.2 0.5 1.3 2.5 3.3 57Slpi Slpi _ Secretory leukocyte protease inhibitor (U73004)(4819) 0.3 0.4 0.8 1.3 2.5 1.7 31Upp Upp _ Uridine phosphorylase (D44464)(7963) -0.1 0.1 0.2 1.1 2.4 2.5 9Scvr Scvr _ Scavenger receptor (L04275)(15614) -0 0.1 0.8 1.3 2.4 1.7 577Ddt Ddt _ D-dopachrome tautomerase (AF012431)(15795) -0.1 0.1 0.3 0.5 2.3 2.3 414Batf Batf _ Basic leucine zipper transcription factor, ATF-like (AF017021)(1727) -0 0.4 0.9 0.9 2.3 1.5 19Klra2 Klra2 _ Killer cell lectin-like receptor, subfamily A, member 2 (U10304)(2699) -0 -0.1 0.3 1 2.2 2.2 6Rcor Rcor _ RE1-silencing transcription factor (REST) co-repressor (AK008448)(3845) -0.1 -0.1 0.2 0.4 2.1 1.7 8Casp1 Casp1 _ Caspase 1 (L28095)(14113) -0.1 -0.1 0.7 1.3 2.1 1.3 373His1a His1a _ Histone H1 (M25365)(6681) 0.2 0 -0.6 -1 2 2.2 571Dlgh3 Dlgh3 _ discs, large homolog 3 (Drosophila) (NM_026580)(14899) 0 -0.1 0.1 0 2 0.6 63Coro1a _ Mus musculus coronin, actin binding protein 1A (Coro1a), mRNA (NM_009898)(10682) 0 -0.1 0.2 0.7 2 1.9 64Nos2 _ Mus musculus nitric oxide synthase 2, inducible, macrophage (Nos2), mRNA (NM_010927)(1150)-0 0.1 0.5 1.1 2 1.2 5Upp _ Mus musculus uridine phosphorylase (Upp), mRNA (NM_009477)(12270) -0 -0.1 0.1 0.7 2 1.8 8Tlr1 Tlr1 _ Toll-like receptor 1 (NM_030682)(14889) -0.2 0.7 0.9 0.7 2 1.1 19Odc Odc _ Ornithine decarboxylase, structural (NM_013614)(14313) 0.2 0.5 0.8 1.1 2 1.9 405Cadps Cadps _ Ca<2+>dependent activator protein for secretion (D86214)(12649) -0 -0.1 0.2 0.7 1.9 1.8 54Anxa3 Anxa3 _ Annexin A3 (AJ001633)(18333) 0 0.3 0.8 1.2 1.9 1.3 44Kdt1 Kdt1 _ Kidney cell line derived transcript 1 (U13371)(15618) -0.1 -0.2 0.2 0.2 1.8 0.8 121Api6 Api6 _ Apoptosis inhibitory 6 (AF018268)(4847) -0.1 0 -0.1 -0.1 1.8 2.6 13Hist2 Hist2 _ Histone gene complex 2 (Z30940)(12663) 0.1 0.1 0.2 0.5 1.8 1.1 19Myo5b Myo5b _ myosin Vb (AK010042)(2468) 0.2 0.1 0.6 1.1 1.8 0.3 88Ltb4r1 Ltb4r1 _ leukotriene B4 receptor 1 (AF077673)(16649) 0.1 0.1 -0.1 -0.1 1.7 1.4 6Mmp14 Mmp14 _ Matrix metalloproteinase 14 (membrane-inserted) (U54984)(4290) 0.4 0.3 0.4 0.2 1.7 1.7 5Barx2 Barx2 _ BarH-like homeobox 2 (AK007378)(2345) -0 0.1 0.1 1.1 1.7 1.1 65Mmp13 Mmp13 _ Matrix metalloproteinase 13 (X66473)(9557) 0.4 0.2 0.4 0.3 1.7 0.6 9Hunk Hunk _ Hormonally upregulated Neu-associated kinase (AF055919)(325) 0.1 0.1 0.4 1 1.7 1.1 35Acp5 Acp5 _ Acid phosphatase 5, tartrate resistant (M99054)(12820) 0.1 0.2 0.4 0.4 1.7 1.4 12Ptgerep2 _ Mus musculus prostaglandin E receptor EP2 subtype (Ptgerep2), mRNA (NM_008964)(4461)-0 -0 -0.1 -0.2 1.7 0.9 4Ptpn14 _ Mus musculus protein tyrosine phosphatase, non-receptor type 14 (Ptpn14), mRNA (NM_008976)(18403)0.1 0.2 0 0.2 1.7 0.7 10SNK _ Mus musculus (clone 2) serum inducible kinase (SNK) mRNA, mRNA sequence (M96163)(7597)0.4 0.1 0.1 0.5 1.6 1.7 6Zfp261 Zfp261 _ zinc finger protein 261 (AK012129)(6907) -0.2 0.1 0 0.1 1.6 1 5C5r1 C5r1 _ Complement component 5, receptor 1 (NM_007577)(533) 0.4 0.8 0.9 0.7 1.6 0 8Brca2 _ Mus musculus breast cancer 2 (Brca2), mRNA (NM_009765)(6064) 0 0.4 0.8 1.1 1.6 0.7 9Folbp1 Folbp1 _ Folate binding protein 1 (AF096319)(7849) -0.1 0 0.1 0 1.6 1.2 20Parn Parn _ poly(A)-specific ribonuclease (deadenylation nuclease) (BC003847)(15215) -0 0 -0.1 -0.1 1.6 2.3 2

4. Late Responses (16hr)

0

50

100

150

200

250

300

1hr 2hr 4hr 8hr 16hr 48hr

Time

Num

ber

of E

lem

ent (

> 3

fold

)

Increase

Decrease

5. Tumor Necrosis Factor-related TranscriptsSymbol Gene Name 1hr 2hr 4hr 8hr 16hr 48hr Copy

Tnf Tnf _ Mus musculus tumor necrosis factor (Tnf), mRNA (NM_013693)(12240) 2.49 2.18 2.09 1.96 1.67 1.00 169Tnf Tnf _ tumor necrosis factor (M38296)(4173) 2.08 1.94 1.89 1.60 1.51 0.88 432Tnf Tnf _ Tumor necrosis factor (X02611)(15709)_TNF ALPHA, TNFSF2, Tnfsf1a 2.74 2.33 2.26 2.11 1.82 1.00 149Tnfaip1 Tnfaip1 _ Tumor necrosis factor, alpha-induced protein 1 (endothelial) (NM_009395)(1828) 0.12 0.07 0.11 0.16 0.21 0.01 349Tnfaip2 Tnfaip2 _ Mus musculus tumor necrosis factor, alpha-induced protein 2 (Tnfaip2), mRNA (NM_009396)(9158)1.40 2.17 1.65 0.64 1.54 0.37 39Tnfaip2 Tnfaip2 _ Tumor necrosis factor, alpha-induced protein 2 (L24118)(6460) 1.38 2.14 1.74 0.63 1.40 0.37 244Tnfaip3 Tnfaip3 _ Mus musculus tumor necrosis factor, alpha-induced protein 3 (Tnfaip3), mRNA (NM_009397)(2887)1.73 2.01 1.71 1.02 1.28 0.61 33Tnfip6 Tnfip6 _ Tumor necrosis factor induced protein 6 (U83903)(12634) 0.20 0.20 0.25 0.43 0.56 0.40 7Tnfrsf11a Tnfrsf11a _ Tumor necrosis factor receptor superfamily, member 11a (AF019046)(12770) -0.13 0.11 -0.03 0.26 0.69 -0.04 21Tnfrsf11b Tnfrsf11b _ Tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) (AB013898)(17434)0.03 0.16 -0.05 -0.08 -0.02 -0.26 7Tnfrsf12a Tnfrsf12a _ tumor necrosis factor receptor superfamily, member 12a (AK005449)(13217) -0.29 -0.59 0.70 1.07 0.61 0.78 73Tnfrsf13b Tnfrsf13b _ Tumor necrosis factor receptor superfamily, member 13b (AF257673)(9939) -0.05 -0.16 -0.16 -0.15 0.85 1.00 17Tnfrsf17 Tnfrsf17 _ Tumor necrosis factor receptor superfamily, member 17 (AF061505)(4985) -0.03 -0.01 -0.05 0.09 -0.33 -0.96 11Tnfrsf18 Tnfrsf18 _ Tumor necrosis factor receptor superfamily, member 18 (AF229433)(7947) 0.18 0.05 0.15 -0.03 0.01 -0.03 4Tnfrsf19 Tnfrsf19 _ Tumor necrosis factor receptor superfamily, member 19 (AF167552)(573) -0.04 0.04 -0.02 -0.02 -0.05 0.13 6Tnfrsf1a Tnfrsf1a _ Tumor necrosis factor receptor superfamily, member 1a (NM_011609)(560) 0.15 0.22 0.56 0.62 0.09 -0.08 309Tnfrsf1a Tnfrsf1a _ Tumor necrosis factor receptor superfamily, member 1a (X57796)(12618) 0.05 0.20 0.62 0.72 0.12 -0.07 353Tnfrsf1al1 Tnfrsf1al1-pending _ Tumor necrosis factor receptor superfamily, member 1a-like 1 (NM_024290)(16583)0.12 -0.02 0.10 0.53 0.47 0.26 50Tnfrsf1al2 Tnfrsf1al2-pending _ Tumor necrosis factor receptor superfamily, member 1a-like 2 (AK012838)(18169)0.01 -0.12 0.03 0.36 0.12 0.34 49Tnfrsf1b Tnfrsf1b _ Tumor necrosis factor receptor superfamily, member 1b (M59378)(10944) 0.26 1.04 1.44 1.32 2.34 1.53 49Tnfrsf20 Tnfrsf20-pending _ Tumor necrosis factor receptor superfamily, member 20 (AB040432)(11359)0.05 0.09 0.03 0.17 -0.12 -0.15 6Tnfrsf22 Tnfrsf22 _ Mus musculus mRNA for voltage-gated sodium channel alpha subunit (Scn4a gene) (AJ278787)(2630)0.08 -0.05 -0.07 -0.02 -0.13 0.03 10Tnfrsf23 Tnfrsf23 _ tumor necrosis factor receptor superfamily, member 23 (AK013022)(10344) 0.04 0.05 0.08 0.17 0.04 0.02 312Tnfrsf4 Tnfrsf4 _ Tumor necrosis factor receptor superfamily, member 4 (X85214)(15779) 0.07 0.05 0.16 -0.11 -0.11 0.02 10Tnfrsf5 Tnfrsf5 _ Tumor necrosis factor receptor superfamily, member 5 (M83312)(9525)_CD40, CD40L RECEPTOR, CD40L, TRAP, p502.19 2.57 3.48 3.76 2.11 1.62 14Tnfrsf6 Tnfrsf6 _ Mus musculus, Similar to ADP-ribosylation-like 4, clone MGC:5774, mRNA, complete cds (BC003725)(4147)-0.01 0.04 -0.02 0.06 -0.05 0.07 11Tnfrsf7 Tnfrsf7 _ Tumor necrosis factor receptor superfamily, member 7 (L24495)(41) -0.02 0.02 0.00 0.06 -0.20 0.07 12Tnfrsf8 Tnfrsf8 _ Tumor necrosis factor receptor superfamily, member 8 (U25416)(11158) -0.09 -0.22 0.13 0.06 0.15 -0.15 6Tnfrsf9 Tnfrsf9 _ Tumor necrosis factor receptor superfamily, member 9 (J04492)(2144) -0.02 0.12 -0.01 0.09 -0.13 -0.01 25Tnfsf10 Tnfsf10 _ Tumor necrosis factor (ligand) superfamily, member 10 (U37522)(7935) 0.11 0.04 0.04 0.03 -0.04 -0.03 7Tnfsf11 Tnfsf11 _ Tumor necrosis factor (ligand) superfamily, member 11 (AF053713)(8911) NA 0.24 -0.12 -0.29 0.25 0.11 2Tnfsf12 Tnfsf12 _ Tumor necrosis factor (ligand) superfamily, member 12 (AF030100)(6475) 0.05 0.05 0.04 0.05 -0.06 -0.02 8Tnfsf12 Tnfsf12 _ tumor necrosis factor (ligand) superfamily, member 12 (AK003722)(939) 0.03 -0.03 -0.18 -0.60 -1.14 -0.76 50Tnfsf14 Tnfsf14 _ Tumor necrosis factor (ligand) superfamily, member 14 (AB029155)(16170) 0.01 0.00 -0.25 -0.48 -0.93 -0.74 18Tnfsf19 Tnfsf19-pending _ Tumor necrosis factor (ligand) superfamily, member 19 (AJ011386)(16040)0.12 0.27 0.12 0.21 0.05 -0.02 66Tnfsf4 Tnfsf4 _ Tumor necrosis factor (ligand) superfamily, member 4 (U12763)(6528) 0.10 0.25 0.13 0.06 0.01 -0.14 6Tnfsf5 Tnfsf5 _ Tumor necrosis factor (ligand) superfamily, member 5 (X65453)(8022) -0.05 -0.02 0.11 -0.08 -0.05 -0.05 10Tnfsf7 Tnfsf7 _ Tumor necrosis factor (ligand) superfamily, member 7 (Y13636)(5019) 0.01 -0.03 0.01 -0.03 0.01 0.27 6Tnfsf8 Tnfsf8 _ Tumor necrosis factor (ligand) superfamily, member 8 (L09754)(6430) -0.12 -0.04 0.02 -0.05 0.09 -0.01 17

6. Interleukin-related Transcripts

Symbol Gene Name 1hr 2hr 4hr 8hr 16hr 48hr CopyIl10 Il10 _ Interleukin 10 (NM_010548)(3225) 1.78 1.98 1.08 1.63 1.32 1.78 16IL10 IL10 _ Mus musculus interleukin 10 (Il10), mRNA (NM_010548)(4474) 1.87 2.14 1.28 1.76 1.40 1.96 8Il10ra Il10ra _ Interleukin 10 receptor, alpha (L12120)(11274) 0.30 0.41 0.56 0.36 0.02 -0.21 13Il10rb Il10rb _ Interleukin 10 receptor, beta (U53696)(14068) 0.27 0.30 0.10 -0.13 0.46 0.27 173Il11 Il11 _ Interleukin 11 (U03421)(14496) 0.06 -0.06 -0.11 -0.04 0.25 1.39 5Il11ra1 Il11ra1 _ interleukin 11 receptor, alpha chain 1 (AK017844)(8747) 0.00 -0.25 -0.01 -0.16 0.86 1.08 18Il11ra2 Il11ra2 _ Interleukin 11 receptor, alpha chain 2 (NM_010550)(5521) 0.01 -0.13 0.00 0.00 0.24 0.18 13Il12a Il12a _ Interleukin 12a (M86672)(8403) 0.04 0.00 -0.03 -0.02 -0.12 -0.04 9Il12b Il12b _ Interleukin 12b (M86671)(1571) 0.02 0.00 0.04 0.08 0.06 0.17 8Il12rb1 Il12rb1 _ interleukin 12 receptor, beta 1 (AK014261)(5301) 0.15 -0.02 -0.03 0.06 0.13 0.07 7Il12rb2 Il12rb2 _ Interleukin 12 receptor, beta 2 (U64199)(14299) -0.01 0.02 0.15 0.00 0.02 -0.12 5Il13 Il13 _ Interleukin 13 (M23504)(6349) 0.19 0.14 0.05 0.24 0.04 0.04 8Il13ra1 Il13ra1 _ interleukin 13 receptor, alpha 1 (S80963)(8914) 0.08 0.25 0.75 1.22 1.11 0.33 181Il13ra2 Il13ra2 _ Interleukin 13 receptor, alpha 2 (U65747)(9675) 0.37 0.78 1.69 2.44 1.71 0.38 9Il15 Il15 _ Interleukin 15 (U14332)(9551) -0.01 0.17 1.06 1.21 1.26 1.68 10Il15ra Il15ra _ Interleukin 15 receptor, alpha chain (U22339)(15937) -0.32 -0.32 0.63 0.97 1.50 1.14 12Il16 Il16 _ Interleukin 16 (AF175292)(4914) -0.49 -0.62 0.02 -0.27 -0.40 -1.37 105Il17 Il17 _ Interleukin 17 (U43088)(6435) -0.05 -0.02 0.00 -0.04 0.12 0.20 10Il17br Il17br _ IL-17B receptor (AF208109)(16175) -0.07 -0.04 0.09 -0.05 0.16 -0.09 6Il17r Il17r _ Interleukin 17 receptor (U31993)(287) -0.18 -0.27 -0.17 -0.34 -0.46 -0.41 472Il18 Il18 _ Interleukin 18 (D49949)(7848) -0.08 0.05 1.27 2.07 1.44 1.18 196Il18r1 Il18r1 _ interleukin 18 receptor 1 (AK017362)(2361) -0.07 -0.08 -0.09 -0.19 0.02 -0.11 117Il18rap Il18rap _ Interleukin 18 receptor accessory protein (AF077347)(11138) 0.13 0.16 0.20 -0.04 0.29 0.05 13Il1a Il1a _ Interleukin 1 alpha (X01450)(16708) 1.45 2.62 3.18 2.10 4.47 3.09 9IL1a IL1a _ Mus musculus interleukin 1 alpha (Il1a), mRNA (NM_010554)(2818) 1.45 2.46 3.13 2.00 4.36 3.14 13Il1b Il1b _ Interleukin 1 beta (M15131)(3465) 2.94 4.36 4.62 4.04 5.05 3.79 39Il1b Il1b _ Interleukin 1 beta (NM_008361)(11552) 2.78 3.94 4.03 3.65 4.28 3.42 75IL1b IL1b _ Mus musculus interleukin 1 beta (Il1b), mRNA (NM_008361)(1321)_IL-1, IL1F2 1.91 3.64 3.87 3.27 4.55 3.36 16Il1f5 Il1f5 _ interleukin 1 family, member 5 (delta) (NM_026124)(10073) 0.03 -0.03 0.01 0.24 0.03 0.13 433Il1f6 Il1f6 _ interleukin 1 family, member 6 (AK004785)(11861) 0.20 0.18 0.20 0.36 0.53 0.46 324Il1hy1 Il1hy1-pending _ Interleukin 1 receptor antagonist homolog 1 (AJ250429)(11246) 0.19 0.13 0.11 0.01 0.04 -0.07 10Il1r1 Il1r1 _ Interleukin 1 receptor, type I (M20658)(9466) 0.05 -0.08 0.05 0.01 -0.17 -0.29 16Il1r2 Il1r2 _ Interleukin 1 receptor, type II (X59769)(9503) 0.25 0.10 0.00 -0.08 -0.25 0.05 8Il1rak Il1rak _ Interleukin 1 receptor-associated kinase (AF103876)(17621) 0.00 0.01 -0.02 -0.12 -0.39 -0.51 137Il1rap Il1rap _ Interleukin 1 receptor accessory protein (X85999)(15953) 0.03 -0.02 0.46 0.57 0.89 0.57 23Il1rapl2 Il1rapl2 _ interkeukin 1 receptor accessory protein-like 2 (NM_023597)(10529) 0.02 -0.13 -0.13 -0.19 -0.38 NA 5Il1rl1 Il1rl1 _ interleukin 1 receptor-like 1 (NM_025319)(5559) -0.05 -0.09 -0.04 -0.02 -0.01 0.04 128Il1rn Il1rn _ Interleukin 1 receptor antagonist (M64404)(6346) 1.45 1.98 3.16 3.95 4.36 2.56 172Il1rrp Il1rrp _ Interleukin 1 receptor related protein (U43673)(6496) -0.03 0.04 0.04 0.02 -0.04 -0.18 8Il2 Il2 _ Interleukin 2 (X01772)(4320) 0.17 -0.14 -0.03 -0.12 -0.08 -0.04 4Il21 Il21 _ Interleukin 21 (NM_021782)(13555) 0.23 0.08 -0.06 -0.05 -0.16 -0.23 7Il21r Il21r _ Mus musculus low density lipoprotein receptor related protein LRP1B/LRP-DIT mRNA, complete cds (AF270884)(18135)-0.11 0.05 0.02 -0.09 -0.07 -0.13 8Il23a Il23a _ Interleukin 23, alpha subunit p19 (NM_031252)(2758) 0.04 0.02 -0.04 -0.01 0.13 -0.04 39Il25 Il25 _ interleukin 25 (AK005126)(682) 0.02 -0.01 -0.11 -0.35 -0.45 -0.33 245Il2ra Il2ra _ Interleukin 2 receptor, alpha chain (M30856)(10499) -0.01 -0.01 -0.03 -0.12 0.14 0.20 6Il2rb Il2rb _ Interleukin 2 receptor, beta chain (NM_008368)(7839) 0.00 0.02 0.11 -0.01 0.11 0.09 13Il2rg Il2rg _ Interleukin 2 receptor, gamma chain (U21795)(9506) 0.27 0.40 0.53 0.55 0.70 0.37 171Il6 Il6 _ Interleukin 6 (J03783)(7872) 0.07 1.14 2.81 3.26 5.11 3.81 14Il6ra Il6ra _ Interleukin 6 receptor, alpha (X53802)(9383) -0.10 -0.28 -0.38 -0.17 -0.54 -0.58 455Il6st Il6st _ Interleukin 6 signal transducer (M83336)(283) -0.01 -0.23 -0.23 -0.06 0.13 -0.38 35Il3 Il3 _ Interleukin 3 (X02732)(17261) 0.01 0.12 0.09 0.05 -0.06 -0.21 10Il3ra Il3ra _ Interleukin 3 receptor, alpha chain (X64534)(15241) 0.09 0.06 0.01 0.09 -0.14 -0.23 26Il4 Il4 _ Interleukin 4 (M25892)(4751) 0.07 0.00 -0.01 -0.14 -0.17 -0.07 10Il4i1 Il4i1 _ Mus musculus, Similar to MBIP protein, clone MGC:7681, mRNA, complete cds (BC002277)(2202)0.02 0.04 -0.01 -0.09 0.12 0.26 270Il4ra Il4ra _ Interleukin 4 receptor, alpha (M27960)(10931) 0.02 0.07 0.09 0.09 1.09 0.47 28Il5 Il5 _ Interleukin 5 (X06271)(6465) 0.15 0.14 -0.01 -0.05 0.04 0.02 6Il5ra Il5ra _ Interleukin 5 receptor, alpha (D90205)(30) -0.13 0.20 0.12 0.18 0.00 0.02 20Il7 Il7 _ Interleukin 7 (X07962)(17252) 0.01 0.13 0.32 0.72 0.12 0.20 8Il7r Il7r _ Interleukin 7 receptor (M29697)(17231) 0.63 0.51 0.11 0.31 0.56 0.49 8Il9 Il9 _ interleukin 9 (AK009243)(7016) -0.22 -0.46 -0.52 -0.52 -0.57 -0.04 84Il9r Il9r _ Interleukin 9 receptor (M84746)(10991) 0.31 -0.04 0.07 0.23 -0.05 0.00 5Ilf3 Ilf3 _ Interleukin enhancer binding factor 3 (AF098967)(12799) 0.09 0.07 -0.11 -0.33 -0.23 -0.44 509Ilk Ilk _ Integrin linked kinase (U94479)(4916) 0.06 0.05 0.05 0.06 -0.19 -0.33 781Iltif Iltif _ Interleukin 10-related T cell-derived inducible factor (AJ249491)(6719) -0.13 -0.02 -0.08 0.02 -0.04 -0.06 8

7. Interferon-related Transcripts

Symbol Gene Name 1hr 2hr 4hr 8hr 16hr 48hr CopyIfi1 Ifi1 _ Interferon inducible protein 1 (U19119)(3539) -0.22 0.85 3.24 3.23 2.04 2.32 384Ifi202a Ifi202a _ Interferon activated gene 202A (M31418)(12729) 0.09 0.58 4.67 6.24 5.84 5.15 27Ifi202b Ifi202b _ Interferon activated gene 202B (NM_011940)(14550) 0.10 NA 4.90 6.14 5.87 5.17 36Ifi203 Ifi203 _ Interferon activated gene 203 (AF022371)(4990) -0.09 -0.03 2.13 3.00 2.09 2.11 15Ifi204 Ifi204 _ Interferon activated gene 204 (M31419)(12878) 0.34 0.68 3.60 4.72 4.07 3.62 53Ifi47 Ifi47 _ Interferon gamma inducible protein, 47 kDa (M63630)(12833) -0.16 -0.37 1.69 2.57 0.73 1.48 153Ifit1 Ifit1 _ Interferon-induced protein with tetratricopeptide repeats 1 (U43084)(1788) 0.29 1.89 4.00 4.05 3.11 2.85 28Ifit2 Ifit2 _ Interferon-induced protein with tetratricopeptide repeats 2 (U43085)(14057) -0.34 0.39 4.32 4.69 4.34 3.29 104Ifit3 Ifit3 _ Interferon-induced protein with tetratricopeptide repeats 3 (L32974)(17199) -0.08 1.37 4.87 5.25 4.55 4.12 42Ifitm3l Ifitm3l _ interferon induced transmembrane protein 3-like (AK014405)(8869) -0.02 -0.21 0.05 0.26 0.17 0.20 569Ifitml Ifitml-pending _ Interferon induced transmembrane protein, like (NM_030694)(5339)0.12 0.15 0.34 0.46 0.45 0.40 752Ifna1 Ifna1 _ Interferon alpha family, gene 1 (X01974)(9897) 0.13 -0.05 0.10 0.18 0.16 0.06 12Ifna11 Ifna11 _ Interferon alpha family, gene 11 (M68944)(9663) 0.13 0.16 -0.05 0.12 -0.01 -0.19 5Ifna11 Ifna11 _ Mus musculus, Similar to CG6385 gene product, clone MGC:6279, mRNA, complete cds (BC003456)(16578)-0.01 -0.13 0.03 -0.10 -0.01 -0.09 10Ifna2 Ifna2 _ Interferon alpha family, gene 2 (K01411)(302) -0.09 0.17 0.06 0.10 0.10 0.00 6Ifna2 Ifna2 _ Interferon alpha family, gene 2 (NM_010503)(2310) -0.07 0.09 -0.05 0.10 0.16 0.02 9Ifna4 Ifna4 _ Interferon alpha family, gene 4 (X01973)(13017) 0.07 -0.12 0.03 0.11 0.16 -0.12 10Ifna5 Ifna5 _ Interferon alpha family, gene 5 (X01971)(16137) 0.01 0.05 0.03 0.06 0.22 -0.14 9Ifna5 Ifna5 _ Mouse gene fragment for delta-immunoglobulin encoding one of two alternate C-termini (exon 8?) (V00789)(598)-0.38 0.11 0.24 0.18 -0.31 0.11 19Ifna6 Ifna6 _ Interferon alpha family, gene 6 (X01972)(3730) 0.06 0.04 0.05 0.16 0.09 -0.02 11Ifna7 Ifna7 _ Interferon alpha family, gene 7 (M13710)(6704) 0.16 0.06 0.02 0.12 0.16 0.03 7Ifna7 Ifna7 _ Mouse alpha leukocyte interferon gene, complete cds (M28587)(465) 0.20 0.06 0.05 0.08 -0.02 -0.14 5Ifna8 Ifna8 _ Interferon alpha family, gene 8 (D00460)(167) -0.01 0.09 0.00 0.12 -0.02 -0.04 4Ifna9 Ifna9 _ Interferon alpha family, gene 9 (M13660)(3584) 0.02 -0.08 -0.06 -0.02 -0.26 -0.05 10Ifnab Ifnab _ Interferon alpha family, gene B (L38698)(2024) 0.07 0.09 0.01 0.08 0.15 -0.05 10Ifnar Ifnar _ Interferon (alpha and beta) receptor (M89641)(11024) 0.02 -0.04 -0.07 0.02 0.45 0.52 270Ifnar2 Ifnar2 _ Interferon (alpha and beta) receptor 2 (Y09865)(8091) -0.42 NA -0.13 -0.32 0.41 0.12 28Ifnb Ifnb _ Interferon beta, fibroblast (NM_010510)(12557) 3.00 3.58 3.94 4.30 3.07 0.59 9Ifng Ifng _ Interferon gamma (M28621)(1572) -0.04 0.02 0.02 0.02 -0.06 0.04 9Ifngr Ifngr _ interferon gamma receptor (AK013367)(14907) -0.12 -0.09 -0.11 -0.04 0.36 -0.11 24Ifngr Ifngr _ Interferon gamma receptor (M28233)(10932) -0.09 -0.23 -0.38 -0.51 -0.37 -0.12 405Ifngr2 Ifngr2 _ interferon gamma receptor 2 (S69336)(15183) 0.07 0.30 0.27 0.18 0.34 0.15 327Ifrd1 Ifrd1 _ Interferon-related developmental regulator 1 (NM_013562)(9463) 0.42 0.33 0.19 0.22 1.55 0.90 1006Ifrd2 Ifrd2 _ interferon-related developmental regulator 2 (AK008144)(10518) -0.06 0.04 0.07 -0.03 0.19 0.07 4Ifrd2 Ifrd2 _ Interferon-related developmental regulator 2 (NM_025903)(7399) 0.04 0.06 -0.02 -0.06 0.03 -0.09 5Ifrg15 Ifrg15-pending _ Interferon alpha responsive gene, 15 kDa (NM_022329)(16328) 0.00 0.27 1.31 1.65 0.26 0.61 1405Irf1 Irf1 _ Interferon regulatory factor 1 (M21065)(15677) 0.44 1.02 2.21 1.92 0.52 0.70 29Irf2 Irf2 _ Interferon regulatory factor 2 (J03168)(4786) 0.02 0.06 0.14 0.12 -0.02 0.00 306Irf3 Irf3 _ Interferon regulatory factor 3 (U75839)(9450) 0.02 -0.02 -0.05 -0.03 0.02 -0.11 232Irf4 Irf4 _ interferon regulatory factor 4 (NM_026010)(13347) -0.50 -0.10 -0.24 -0.47 -0.84 -0.57 139Irf5 Irf5 _ Interferon regulatory factor 5 (AF028725)(18271) 0.05 0.34 0.17 0.64 0.40 0.13 59Irf5 Irf5 _ interferon regulatory factor 5 (AK012400)(17992) 0.00 0.14 0.04 -0.03 0.04 -0.04 19Irf6 Irf6 _ Interferon regulatory factor 6 (U73029)(7891) -0.06 -0.17 -0.21 -0.33 -0.41 -0.18 13Irf7 Irf7 _ Interferon regulatory factor 7 (U73037)(14096) 0.01 0.41 1.99 2.71 2.46 2.83 213

9. Bio-Plex Cytokine Antibody Bead Experiments (Performed by Ruth Levitz in the Antibody Lab)

Time Course of TNF-α Secretion in Response to Ligand Stimulation

0.0

9000.0

18000.0

27000.0

0 1 2 3 4 5

Time (Hours)

pg/m

l

Untreated

LPS

IFG

IFG + LPS

IL1b

IL1a

IL6

IL10

G-CSF

IL12(p70)

MIP-1a

RANTES

TNFa

LPS

Ingenuity Pathway Analysis

11. LPS Network

IL1a, IL1bTNF

TNFRSF5

TNFAIP3

Products: Ap2, Birc2, C3, CD83, Clecsf9, Csf3, Dusp2, Egr1, Ier2, Ier3, Ifnb, IL10, IL1b, Planh1, Ptpn16, Saa3, Scya4, Scyb10, Scyb2, Slfn2, Tnf, Tnfaip3, Tnfrsf5, Traf1, Zfp36, ……

10. LPS Stimulated NF-kB Pathway

Summaries of LPS Signaling

8. Cytokines Targeted by Bio-Plex

1.001.822.112.262.332.74Tnf _ Tumor necrosis factor _15709_X02611

1.001.671.962.092.182.49Tnf _ Mus musculus tumor necrosis factor (Tnf) _12240_NM_013693TNF-alpha

3.263.663.092.932.200.86Ccl5 _ Mus musculus small inducible cytokine A5 (Scya5), NM_013653RANTES

N/AN/AN/AN/AN/AN/ACcl3MIP-1alpha

N/AN/AN/AN/AN/AN/AKerotinocyte cytokineKC

0.04-0.060.020.020.02-0.04Ifng _ Interferon gamma _1572_M28621IFN-gamma

0.300.950.540.540.610.16Csf2 _ Colony stimulating factor 2 (granulocyte-macrophage)_X03019GM-CSF

2.704.413.493.402.912.66Csf3 _ Colony stimulating factor 3 (granulocyte) _6317_NM_009971G-CSF

0.200.12-0.040.00-0.02-0.05Il17 _ Interleukin 17 _6435_U43088IL-17

0.170.050.080.040.000.02Il12b _ Interleukin 12b _1571_M86671IL-12 (p40)

0.140.260.270.290.190.35Il12a _ Mus musculus interleukin 12 p35 subunit, M86672

-0.04-0.12-0.02-0.030.000.00Il12a _ Interleukin 12a _8403_M86672IL-12 (p35)

1.961.401.761.282.141.87Il10 _ Mus musculus interleukin 10 (Il10)_4474_NM_010548

1.781.321.631.081.981.78Il10 _ Interleukin 10 _3225_NM_010548IL-10

3.815.113.262.811.140.07Il6 _ Interleukin 6 _7872_J03783IL-6

-0.04-0.050.170.090.030.06Il5 _ Mus musculus interleukin 5 (Il5), mRNA _18419_NM_010558IL-5

-0.07-0.17-0.14-0.010.000.07Il4 _ Interleukin 4 _4751_M25892IL-4

0.01-0.010.05-0.11-0.03-0.01Il3 _ Mus musculus interleukin 3 (Il3), mRNA _2852_NM_010556IL-3

-0.11-0.280.120.100.04-0.01Il2 _ Mus musculus interleukin 2 (Il2), mRNA _2909_NM_008366

-0.04-0.06-0.17-0.03-0.100.15Il2 _ Interleukin 2 _4320_X01772IL-2

3.364.553.233.873.641.91Il1b _ Mus musculus interleukin 1 beta (Il1b), _1321_NM_008361

3.795.054.044.624.362.94Il1b _ Interleukin 1 beta _3465_M15131

3.424.283.654.033.942.78Il1b _ Interleukin 1 beta _11552_NM_008361IL-1beta

3.144.362.003.132.461.45Il1a _ Mus musculus interleukin 1 alpha (Il1a) _2818_NM_010554

3.094.472.103.182.621.21Il1a _ Interleukin 1 alpha _16708_X01450IL-1alpha

48hr16hr8hr4hr2hr1hrGeneCytokines

1.001.822.112.262.332.74Tnf _ Tumor necrosis factor _15709_X02611

1.001.671.962.092.182.49Tnf _ Mus musculus tumor necrosis factor (Tnf) _12240_NM_013693TNF-alpha

3.263.663.092.932.200.86Ccl5 _ Mus musculus small inducible cytokine A5 (Scya5), NM_013653RANTES

N/AN/AN/AN/AN/AN/ACcl3MIP-1alpha

N/AN/AN/AN/AN/AN/AKerotinocyte cytokineKC

0.04-0.060.020.020.02-0.04Ifng _ Interferon gamma _1572_M28621IFN-gamma

0.300.950.540.540.610.16Csf2 _ Colony stimulating factor 2 (granulocyte-macrophage)_X03019GM-CSF

2.704.413.493.402.912.66Csf3 _ Colony stimulating factor 3 (granulocyte) _6317_NM_009971G-CSF

0.200.12-0.040.00-0.02-0.05Il17 _ Interleukin 17 _6435_U43088IL-17

0.170.050.080.040.000.02Il12b _ Interleukin 12b _1571_M86671IL-12 (p40)

0.140.260.270.290.190.35Il12a _ Mus musculus interleukin 12 p35 subunit, M86672

-0.04-0.12-0.02-0.030.000.00Il12a _ Interleukin 12a _8403_M86672IL-12 (p35)

1.961.401.761.282.141.87Il10 _ Mus musculus interleukin 10 (Il10)_4474_NM_010548

1.781.321.631.081.981.78Il10 _ Interleukin 10 _3225_NM_010548IL-10

3.815.113.262.811.140.07Il6 _ Interleukin 6 _7872_J03783IL-6

-0.04-0.050.170.090.030.06Il5 _ Mus musculus interleukin 5 (Il5), mRNA _18419_NM_010558IL-5

-0.07-0.17-0.14-0.010.000.07Il4 _ Interleukin 4 _4751_M25892IL-4

0.01-0.010.05-0.11-0.03-0.01Il3 _ Mus musculus interleukin 3 (Il3), mRNA _2852_NM_010556IL-3

-0.11-0.280.120.100.04-0.01Il2 _ Mus musculus interleukin 2 (Il2), mRNA _2909_NM_008366

-0.04-0.06-0.17-0.03-0.100.15Il2 _ Interleukin 2 _4320_X01772IL-2

3.364.553.233.873.641.91Il1b _ Mus musculus interleukin 1 beta (Il1b), _1321_NM_008361

3.795.054.044.624.362.94Il1b _ Interleukin 1 beta _3465_M15131

3.424.283.654.033.942.78Il1b _ Interleukin 1 beta _11552_NM_008361IL-1beta

3.144.362.003.132.461.45Il1a _ Mus musculus interleukin 1 alpha (Il1a) _2818_NM_010554

3.094.472.103.182.621.21Il1a _ Interleukin 1 alpha _16708_X01450IL-1alpha

48hr16hr8hr4hr2hr1hrGeneCytokines

Introduction

Microarray experiments using RAW 264.7 cells treated with LPS (Lipopolysaccharide, 100ng/ml) / LBP (LPS-binding protein, 100pM) (0, 1, 2, 4, 8, 16, and 48hr) were performed. Time matched untreated samples were used as controls. Dye-swapping was also carried out, in which controls were labeled with Cy3 (green dye) and test samples with Cy5 (red) in the 1st run, and vice versa in the 2nd run. This eliminates possible bias caused by variation in incorporation efficiency of the dyes. Samples were prepared in triplicate by The Cell Lab (Dallas) and hybridizations were performed on 16K Operon-Compugen Oligonucleotide Arrays. The transcription analysis of LPS/LBP treated RAW cells showed about 70 to 280 elements regulated more than 2 fold, peaking at 16 hours and providing a picture of the temporal pattern of gene expression responses induced by LPS.

Lipopolysaccharide (LPS) is the major constitute of the outer membrane of Gram-negative bacteria. In blood, LPS forms a complex with serum protein LBP (LPS-binding protein). LPS binds cell the surface receptor CD14 to enhance Toll-like receptor 4 (TLR4)-dependent LPS recognition. After TLR4 forms a complex with protein MD-2, the complex is transported to the cell surface. TLR4, part of the Toll-like receptor family, is a membrane protein with an extracellular leucine-rich repeat domain and a cytoplasmic TIR domain. The activation of TLR4 together with the MyD88 adaptor family members, (including MyD88, TIRAP, TRIF, and TRAM) initiates the intracellular response. MyD88 adaptor protein appears to link TLR4 to IRAK4 kinases, also known as the MyD88-dependent pathway. Upon activation of the cells with LPS, the MyD88-dependent pathway leads to IRAK phosphorylation and TRAF6 ubiquitination. Ubiquitinated TRAF6 activates TAK1, resulting in the activation of the NF-kB, p38, and JNK pathways. TIRAP seems to be responsible for the MyD88-independent response but the precise role is unknown. Both TRIF and TRAM link TLR4 to pathways that activate IRF3 and TBK1. Engagement of TLR4 recruits proximal adaptors TRIF and TRAM, leading to phosphorylation and activation of IRF3 and delayed NF-kB activation.

1. Number of Regulated Genes (>3 fold) in RAW by LPS + LBP

Results

Early responses (1h) by LPS stimulation. Early growth response 1 and 2 (EGR1 and 2), lipopolysaccharide-induced molecular possessing ankyrin-repeats (MAIL-pending), and small inducible cytokines such as cxcl2, cxcl10, ccl4 were significantly induced at early time points. Immunoresponsive gene 1 (IRG1), cxcl10 (IP10), and IFNb are early products of IRF3 pathway. IL1b, TNF, Myc, CSF3, and IL10 were also highly upregulated.Intermediate responses (4h). Many genes in this group are interferon-activated, interferon-induced, and interferon-stimulated genes. Interestingly, some G-protein-related genes, such as GBP3 (guanylate nucleotide binding protein 3), GBP1, GBP2, and RGS16 were induced. IL6, STAT1, and STAT2 were also upregulated.Late responses (16h). Late responses include glutathione S-transferase theta 1, Fc receptor IgG low affinity IIb, caspase 1, histone H1, histone gene complex 2, and inducible nitric oxide synthase 2 (iNOS2).

Throughout the time points tested, there were few downregulated genes and the magnitude of downregulation was relatively small.

The copy number of the transcript was roughly measured by comparing the hybridization intensity.

Bio-Plex (Dallas lab) results revealed that cytokines such as IL1a, IL1b, IL6, IL10, IL12, G-CSF, MIP-1a, RANTES, and TNFa were induced. The array data were consistent with LPS-responses of Bio-Plex experiments except IL12, where Bio-Plex tested IL12 p70 dimer was positive whereas array tested p35 and 40 subunits were negative.

The NFkB pathway map with mRNA expression levels shown provides a basis for the hypothesis that the cellular responses in macrophages, through the recognition of LPS, transcriptionally regulate not only the actual production of the cytokine but also the downstream elements resulting in the release of many cytokines and their related products.

1. Transcriptional regulation of pathways was activated by Toll-like receptor 4 (3 to more than 100 fold).

2. Apparent waves of transcription:Immediate early (1hr), Intermediate (4hr), Late (16hr).

3. Families of genes include specific subsets upregulated by LPS: TNF, Interferon, Interleukin, and chemokine.

4. Possible autocrine loops, and other feedback mechanisms to readjust the patterns of signal transduction.