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Stem Factor Programs the Mast Cell Activation Phenotype Tomonobu Ito, Daniel Smrz, Mi-Yeon Jung, Geethani Bandara, Avanti Desai, Sárka Smrzová, Hye Sun Kuehn, This information is current as Michael A. Beaven, Dean D. Metcalfe and Alasdair M. of September 25, 2021. Gilfillan J Immunol 2012; 188:5428-5437; Prepublished online 23 April 2012;

doi: 10.4049/jimmunol.1103366 Downloaded from http://www.jimmunol.org/content/188/11/5428

Supplementary http://www.jimmunol.org/content/suppl/2012/04/24/jimmunol.110336 Material 6.DC1 http://www.jimmunol.org/ References This article cites 52 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/188/11/5428.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Stem Cell Factor Programs the Mast Cell Activation Phenotype

Tomonobu Ito,*,1 Daniel Smrzˇ,*,1 Mi-Yeon Jung,* Geethani Bandara,* Avanti Desai,* Sˇa´rka Smrzˇova´,* Hye Sun Kuehn,* Michael A. Beaven,† Dean D. Metcalfe,* and Alasdair M. Gilfillan*

Mast cells, activated by Ag via Fc«RI, release an array of proinflammatory mediators that contribute to allergic disorders, such as asthma and anaphylaxis. The KIT , (SCF), is critical for mast cell expansion, differentiation, and survival, and under acute conditions, it enhances mast cell activation. However, extended SCF exposure in vivo conversely protects against fatal Ag-mediated anaphylaxis. In investigating this dichotomy, we identified a novel mode of regulation of the mast cell activation phenotype through SCF-mediated programming. We found that mouse bone marrow-derived mast cells chronically exposed to SCF displayed a marked attenuation of Fc«RI-mediated degranulation and production. The hyporesponsive phenotype Downloaded from was not a consequence of altered signals regulating calcium flux or kinase C, but of ineffective cytoskeletal reorganization with evidence implicating a downregulation of expression of the Src kinase Hck. Collectively, these findings demonstrate a major role for SCF in the homeostatic control of mast cell activation with potential relevance to mast cell-driven disease and the development of novel approaches for the treatment of allergic disorders. The Journal of Immunology, 2012, 188: 5428–5437. http://www.jimmunol.org/ ntigen-mediated mast cell (MC) activation, via FcεRI, Little is known about how SCF and other extrinsic factors, or the results in the release of an array of inflammatory combination thereof, may dictate the MC phenotype with regard to A mediators that underlies allergic reactions in atopic responsiveness to Ag and other stimulants. Under acute experi- disease (1, 2). MCs are derived from bone marrow progenitors that mental conditions, SCF is one of several endogenous agents known migrate into the circulation and peripheral tissues where they to potentiate Ag-mediated MC degranulation and cytokine pro- expand and mature under the influence of contained duction (11–13). Nevertheless, in contrast to its acute effects on within the surrounding milieu (3). KIT activation, as a conse- MC activation in vivo (14), it is reported that repetitive s.c. in- quence of binding of its ligand stem cell factor (SCF) produced jection of SCF over a period of 21 d into mice may actually protect within tissues by stromal cells, is critical for the development of against fatal anaphylactic reactions (15). Indeed, at the sites of by guest on September 25, 2021 MCs from bone marrow progenitor cells and their subsequent injection, the MCs exhibited little morphological evidence of de- accumulation, maturation, and survival in tissues (4). Unlike hu- granulation after induction of anaphylaxis via IgE in these mice man MCs (5), mouse MC development and survival in culture are (figure 2 in Ref. 15), suggesting that chronic exposure to SCF may supported by IL-3 in the absence of SCF (6). However, the sup- have a profoundly different impact on MC activation than does plementary presence of SCF markedly enhances the rate of growth short-term exposure. Thus, we investigated the hypothesis that of these cells and is described to skew the development of the cells prolonged exposure of MCs to SCF, as likely occurs in vivo to to that of a serosal/connective tissue phenotype. In contrast, cells maintain MC homeostasis, may lead to transcriptional modifi- grown in IL-3 alone are considered to more resemble the mucosal cations that alter the underlying activation properties of the cells. phenotype based on the types of proteases expressed (7–10). As reported in this article, these studies led us to identify a novel mechanism for the regulation of the extent of MC activation *Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Dis- through SCF-dependent induction of a hyporesponsive phenotype eases, National Institutes of Health, Bethesda, MD 20892; and †Laboratory of Mo- with respect to both cytokine production and degranulation. This lecular Immunology, National , Lung, and Blood Institute, National Institutes of phenotype was not due to downregulation of the expression of Health, Bethesda, MD 20892 either FcεRI or KIT, but it could be explained by an inability of the 1T.I. and D.S. are co-first authors. cells to undergo the cytoskeletal reorganization required for me- Received for publication December 2, 2011. Accepted for publication March 28, 2012. diator release, potentially as a consequence of decreased expres- sion of the Src kinase Hck. These findings reveal that the This work was supported by the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases and the National Heart, Lung, and Blood sensitivity of MCs to IgE/Ag stimulation is highly regulated by Institute within the National Institutes of Health. SCF and presumably other cytokines in the surrounding tissue The array data presented in this article have been submitted to Expression milieu. This may have important implications for understanding Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE35332. how the activation capacity of tissue MCs may be phenotypically Address correspondence and reprint requests to Dr. Alasdair M. Gilfillan, Laboratory modified in health and in disease. of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11C206, 10 Center Drive MSC 1881, Be- thesda, MD 20892-1881. E-mail address: agilfi[email protected] Materials and Methods The online version of this article contains supplemental material. Cell culture and coculture Abbreviations used in this article: BMMC, bone marrow-derived mast cell; cSCF, chronic SCF; GPCR, -coupled ; b-hex, b-; HSA, Experiments on mice were conducted under a protocol approved by the human serum albumin; MC, mast cell; NIH, National Institutes of Health; PKC, Animal Care and Use Committee at National Institutes of Health (NIH). C; PL, ; RT, room temperature; SCF, stem cell factor. Bone marrow-derived MCs (BMMCs) were developed from bone marrow www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103366 The Journal of Immunology 5429 obtained from femurs of C57BL/6 mice (The Jackson Laboratory, Bar Retroviral transduction Harbor, ME), as described (16). Essentially, the cells were cultured for 4–6 wk in media containing mouse rIL-3 (30 ng/ml; PeproTech, Rocky Hill, Retroviral vector pMX-puro with/without the mouse hck cDNA (a kind gift NJ) or a combination of mouse rIL-3 (30 ng/ml) and mouse recombinant from Dr. Toshiaki Kawakami, La Jolla Institute for Allergy and Immu- SCF (unless otherwise indicated: 100 ng/ml) (PeproTech). The cells were nology, San Diego, CA) was transfected into PLAT-E cells (Cell Biolabs, maintained at 37˚C in a humidified incubator gassed with 95% air and 5% San Diego, CA) with FUGENE 6 transfection reagent (Roche Applied Science) in Opti-MEM (Invitrogen). Transfected cells were grown in CO2. The purity of the cultures, as assessed by toluidine blue staining (17) and FcεRIa and KIT expression, was .99%. The NIH 3T3 mouse fibro- DMEM containing FBS (10%) and L-glutamine (4 mM), and the media blast cell line (American Type Culture Collection, Manassas, VA) was were replaced 16–20 h posttransfection with fresh media containing grown or cocultured (18) with BMMCs in the same media as for BMMCs penicillin (100 U/ml) and streptomycin (100 mg/ml). After 24 h, virus but in the absence of IL-3 and SCF. particles in the media were collected by centrifugation (8000 rpm, over- night, 4˚C), and the pellet was resuspended in complete BMMC medium. Cell sensitization, activation, degranulation, and cytokine/ Viral titer was determined using a QuickTiter retrovirus quantitation 7 chemokine release (Cell Biolabs). A total of 0.5–1 3 10 BMMCs (2–3 wk old) was trans- duced with 4.5 3 1010 virus particles in hexadimerthrine bromide (10 mg/ BMMCs were sensitized overnight in cytokine-containing or cytokine-free ml). After 72 h, the medium was replaced with fresh BMMC medium media (as indicated) with mouse anti–DNP-IgE (100 ng/ml; clone SPE-7; with/without SCF, and transduced cells were selected in the presence of Sigma). After sensitization, the cells were processed and activated as de- 1.2 mg/ml for 2 wk. scribed (16). Degranulation after a 30-min activation was monitored by the release of the granule component b-hexosaminidase (b-hex) into the Quantitative real-time PCR supernatants, as described (19), and expressed as a percentage of b-hex released into supernatant. The amount of cytokines released from cells Total RNAwas isolated using an RNeasy Plus mini kit (QIAGEN, Valencia, CA). One microgram of RNA was used for reverse transcription reaction after a 6-h activation was determined by Quantikine ELISA kits (R&D Downloaded from Systems, Minneapolis, MN). To measure cytokine content within the cy- using random hexamers and Superscript III reverse transcriptase (Invi- toplasm, the activated cells were lysed by adding distilled water followed trogen) in a 20-ml reaction. One microliter of the resulting cDNA was used by freezing (1 h)/thawing, the supernatants were collected, and the amount for real-time PCR using an HCK TaqMan gene-expression assay, ac- of cytokines was determined as above. cording to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Cell fractionation, immunoblotting, and intracellular calcium measurement Affymetrix gene chip analysis http://www.jimmunol.org/ Sensitized BMMCs were stimulated with Ag for 2 min at 37˚C, and cell Bone marrow cells were harvested from six 3-mo-old male littermates in a fractionation was performed, as described (16). The cells were lysed as randomized order on a single day, and cells were cultured for 4 wk in either described (20, 21), and were separated by electrophoresis on IL-3 (30 ng/ml) alone or IL-3+SCF (100 ng/ml); cell suspensions were 4–12% NuPage Bis-Tris gels (Invitrogen, Carlsbad, CA) and probed for centrifuged, and each cell pellet was lysed in 600 ml RLT buffer (QIA- 3 immunoreactive proteins using the following protein-specific Abs: b- GEN). Cell lysates were then homogenized for 2 min at 21,000 g using (Sigma), Hck (Santa Cruz Biotechnology, Santa Cruz, CA), and other a QIAshredder homogenizing column (QIAGEN). Each sample lysate phosphoprotein- and protein-specific Abs (Cell Signaling, Beverly, MA). aliquot (100 ml) was combined with 100 ml RLT buffer, 140 ml100% The immunoreactive proteins were visualized by probing with rabbit IgG- ethanol, and 2-ME (0.145 mM). were extracted using an RNeasy specific Ab (Amersham Biosciences, Piscataway, NJ), or mouse IgG Fc- 96 kit (QIAGEN), as described (24), except that each sample was treated specific Ab (Sigma) conjugated with HRP. To monitor the intracellular with 27 U DNase I during the extraction process. RNA concentrations calcium during cell activation, the sensitized BMMCs were pretreated, were determined by spectrophotometry measuring absorbance at 260 and by guest on September 25, 2021 activated, and analyzed, as described (22). 280 nm, and RNA quality was verified on an Agilent 2100 Bioanalyzer using the Pico analysis kit (Agilent Technologies, Palo Alto, CA). The Toluidine blue and alcian blue/safranin staining sample RNA integrity number values ranged from 8.7 to 9.8, with an average of 9.4. Affymetrix GeneChip targets were made by amplification Cytospins of 4-wk-old BMMCs were prepared, fixed, and stained with of 50 ng RNA using the WT-Ovation RNA Amplification System (Nugen, toluidine blue or alcian blue/safranin, as described (17, 23). San Carlos, CA). The resulting amplified single-stranded cDNAs were Flow cytometric analysis of Fc«RI and KIT surface expression purified according to the QIAquick 96-well protocol (QIAGEN), with a modified centrifugation protocol (24). Each sample was quantified using Following blocking of FcgRs with 2.4G2 (BD Pharmingen), cells were A260/280 spectrophotometry (Molecular Devices, Sunnyvale, CA), stained with FITC–anti-FcεRI (eBioscience) and PE–anti-KIT (BD Phar- and the quality was assessed using Agilent’s 2100 Bioanalyzer (Agilent mingen) Abs to examine surface expression of FcεRI and KIT, respec- Technologies). Amplified cDNAs were fragmented and labeled following tively. Cells were incubated for 1 h at 4˚C and, after washing with PBS, the FL-Ovation cDNA Biotin Module V2 protocol (Nugen). the stained cells were analyzed using a FACSCalibur flow cytometer and Hybridization, fluidics, and scanning were performed according to CellQuest software (BD Biosciences). standard Affymetrix protocols (http://www.affymetrix.com). GeneChip Operating Software (GCOS v1.4) was used to convert the image files to F-actin and a- cell intensity data (cel files). All cel files, representing individual samples, were normalized using the scaling method within expression console (EC Sensitized BMMCs were stimulated with DNP-human serum albumin (HSA) v1.1; http://www.Affymetrix.com) and a scaled target of 1250 to produce (10 ng/ml) for the indicated times, fixed with 4% paraformaldehyde/5 mM the analyzed cel files (chp files) along with the report files. The cel files EGTA and EDTA in PBS for 15 min at room temperature (RT) and then were placed into Partek Genomics Suite software (v6.5 6091110; Partek, F-actin (polymeric) was stained with FITC-labeled phalloidin (Sigma) in St. Louis, MO) and quantile normalized to produce the principal com- 2% BSA/0.1% saponin-PBS for 1 h in the dark at RT. The stained cells were ponents analysis graph. An ANOVA was performed within Partek to analyzed using a FACSCalibur flow cytometer and CellQuest software (BD obtain multiple test-corrected p values using the false discovery rate Biosciences), and the data were analyzed using FlowJo software (Tree Star, method at the 0.01 significance level and was combined with fold-change Ashland, OR). Alternatively, the fixed cells were sedimented, rinsed with values, signal confidence (above background), and call consistency (as a 2% BSA/PBS/5 mM EGTA, and fixed to glass slides by cytospin. The at- percent) as calculated using custom Excel templates for each comparison tached cells were then incubated with an anti–a-tubulin Ab (Cell Signaling) (25). The data have been deposited in Omnibus under at a dilution of 1:100 in 0.1% saponin/2% BSA/PBS/5 mM EGTA for 1 h at accession number GSE35332 (http://www.ncbi.nlm.nih.gov/geo/query/ RT. After washing, FITC-labeled phalloidin (Sigma) and Alexa Fluor 568 acc.cgi?token=pngzzescaqwkwta&acc=GSE35332). donkey anti-rabbit IgG (Invitrogen) were added for 40 min at RT. After washing the slides, mounting solution containing DAPI (Invitrogen) was Statistical analysis added, and the stained cells were imaged by fluorescence microscopy using a Zeiss LSM 510 UV confocal microscope, Plan-Apochromat, NA 633/1.4 Data are presented as the mean and SEM. Statistics were analyzed using the oil. The fluorescence intensity was evaluated from the acquired images two-tailed Student t test. Differences were considered significant at p , using ImageJ software. 0.05; n represents the number of preparations from individual mice. 5430 SCF AND MAST CELL ACTIVATION PROGRAMMING

Results surface expression of FcεRI or KIT (Supplemental Fig. 1B, 1C), Prolonged exposure of developing BMMCs to SCF enhances and there were no noticeable differences in the purity, staining IL-3–mediated growth but subsequently inhibits Ag-mediated characteristics, or morphology of the cells cultured in IL-3 or IL-3 degranulation plus SCF (Supplemental Fig. 1D, 1E), other than that cells ex- posed to SCF were more granulated than were cells cultured in IL- BMMCs were cultured in media containing IL-3, SCF, or IL-3 in 3 alone. Further, the histological examination of cells revealed combination with SCF for up to 8 wk. As reported (10), the that, although the cells cultured in IL-3 alone appeared depleted combination of IL-3 and SCF (100 ng/ml) significantly enhanced of granules following Ag challenge, the cSCF-BMMCs did not the number of MCs in culture compared with IL-3 alone (Sup- (Supplemental Fig. 2). These latter data are consistent with the plemental Fig. 1A). Culturing in SCF alone also supported the conclusion that the reduced Ag-mediated degranulation in the growth of mouse BMMCs; however, the yields were inconsistent cSCF-BMMCs was due to a defect in the process of degranulation and significantly lower than those obtained with either IL-3 alone rather than a defect in granule formation. or IL-3 in combination with SCF (data not shown). In contrast to the ability of SCF to enhance Ag-mediated de- Both soluble and membrane-associated SCF downregulate MC granulation on acute exposure (12, 13), cells cultured in IL-3 in the activation presence of SCF throughout the culture period, hereafter termed In addition to its soluble form, SCF exists as a membrane-associ- chronic SCF-cultured (cSCF)-BMMCs, displayed a marked re- ated moiety. Thus, fibroblast feeder layers, expressing membrane- duction in the capacity of Ag to induce degranulation compared bound SCF on the cell surface, support MC culture (26, 27). with the cells grown in IL-3 alone, regardless of whether the cells Therefore, we examined whether membrane-bound SCF, pre- Downloaded from were nonstarved (Fig. 1A) or starved overnight (Fig. 1B) of sented in this manner, has the same effect as does soluble SCF. cytokines prior to Ag challenge. The attenuated degranulation Two-week-old BMMCs, cultured in IL-3 alone, were incubated observed in the cSCF-BMMCs was not a consequence of reduced for an additional 2 wk on a monolayer of NIH 3T3 fibroblasts, and Ag-mediated activation of BMMCs was examined. The cells grown on the feeder layer, although smaller, were densely gran-

ulated and had a similar morphological appearance to the cells http://www.jimmunol.org/ exposed to soluble SCF (Supplemental Fig. 3). As for BMMCs cultured in the presence of soluble SCF, Ag-induced degranulation in the fibroblast-cultured BMMCs was significantly reduced (Fig. 1C). Together, these data support the conclusion that prolonged exposure of developing BMMCs to either soluble or membrane- bound SCF profoundly attenuates FcεRI-mediated degranulation. Prolonged SCF exposure blocks enhanced degranulation

mediated by acute KIT activation but not by PGE2 and by guest on September 25, 2021 adenosine G protein-coupled receptors When added concurrently with Ag, SCF (12) via the -based receptor KIT, and PGE2 or adenosine via G protein- coupled receptors (GPCRs) (16), synergistically enhance Ag- mediated degranulation, albeit by different mechanisms (16, 28). To examine whether the hypoactive phenotype observed in the cSCF-BMMCs was restricted to FcεRI-regulated MC activation, cells starved of cytokines overnight were acutely challenged with SCF, PGE2, or adenosine in the presence of Ag. To further ex- amine whether the hypoactive phenotype was restricted to receptor-operated pathways, we examined degranulation in re- sponse to PMA/ionomycin, which circumvents receptor require- ments, in BMMCs cultured in the absence or presence of SCF. As shown in Fig. 2A and 2B, the ability of acutely added SCF to enhance Ag-mediated degranulation in the cSCF-BMMCs was also attenuated compared with that observed in the cells grown in IL-3 alone. However, the synergistic actions of PGE2 (Fig. 2C, 2D) and adenosine (Fig. 2E, 2F) were minimally affected. In addition, there was no difference in the capacity of PMA/ FIGURE 1. Extended exposure to SCF downregulates FcεRI-mediated ionomycin to elicit a degranulation response in the BMMCs, re- MC degranulation. (A and B) BMMCs were cultured in IL-3 in the pres- gardless of whether they were cultured in SCF (Fig. 2G). Taken ence or absence of SCF for 4 wk. The cells were then nonstarved (A)or together, these data suggest that the ability of cSCF to alter de- B starved ( ) of cytokines overnight and concurrently sensitized with IgE. granulation may be restricted to receptors that signal through ty- The cells were challenged with the indicated concentrations of Ag (DNP- rosine kinases and not those that signal via GPCRs. HSA) for 30 min, and degranulation (b-hex) was determined. (C) Two- week-old BMMCs, incubated in IL-3 alone, were cocultured with or Downregulation of cytokine production by prolonged exposure without NIH 3T3 fibroblasts for 2 additional weeks, and degranulation of to SCF the cytokine-free medium-sensitized cells was determined as in (A) and (B). Data represent mean and SEM [(A) n =4;(B) n =6;(C) n = 5], and In addition to degranulation, MCs generate cytokines (29). differences between IL-3 and IL-3+SCF–cultured cells are indicated. *p , Therefore, we examined whether sustained exposure to SCF 0.05, t test. downregulated the production of specific cytokines in the same The Journal of Immunology 5431 Downloaded from http://www.jimmunol.org/

FIGURE 3. Extended exposure to SCF downregulates MC cytokine production. (A–E) BMMCs were cultured in IL-3 in the absence (black

bars) or presence (white bars) of SCF for 4 wk, as indicated. The IgE- by guest on September 25, 2021 sensitized cells in cytokine-free media were then challenged for 6 h with

Ag (DNP-HSA) concomitantly with SCF (A–D) or PGE2 (E), and the amount of cytokines released into the media was determined. Data rep- resent mean and SEM [(A, E) n =5;(B–D) n = 3], and differences between IL-3 and IL-3+SCF–cultured cells are indicated. *p , 0.05, t test.

FIGURE 2. Differential effects of extended exposure to SCF on KIT and with Ag to stimulate the production of IL-6 in the cells cultured in GPCR-enhanced MC degranulation. (A–G) BMMCs were cultured in IL-3 the presence of SCF (Fig. 3E), again suggesting that this GPCR in the presence or absence of SCF for 4 wk. The cells were then starved of can overcome the defect induced by extended SCF ex- cytokines overnight and concurrently sensitized with IgE. The cells were posure. challenged with the indicated concentrations of Ag (10 ng/ml) concomi- To verify that the diminution in cytokine release accurately A B C D tantly with SCF [0–100 ng/ml; ( , )], PGE2 [0–100 nM; ( , )], aden- reflected a reduction in cytokine production rather than retention E F osine [0–10,000 nM; ( , )], or PMA/ionomycin [20 nM PMA/100 nM within SCF-treated cells (30), intracellular and extracellular IL-6 G A C E G ionomycin; ( )] for 30 min, degranulation ( , , , ) was determined, and levels were determined in cell lysates and media, respectively. The potentiation, expressed as net b-hex release (B, D, F), was evaluated. Data data showed that relatively little IL-6 was retained within cells represent mean and SEM [(A, B) n =4;(C, D) n =5;(E, F) n = 3], and differences between IL-3 and IL-3+SCF–cultured cells are indicated. after their activation (Supplemental Fig. 3C, 3D; note different *p , 0.05, t test. scales). Thus, the defective cytokine production observed in the cSCF-BMMCs was largely a consequence of a decrease in cyto- kine generation. manner as did degranulation. As shown in Fig. 3, the production of IL-6 (Fig. 3A), TNF-a (Fig. 3B), and, although not statistically Transformation to a hyporesponsive phenotype occurs in significantly, IL-13 (Fig. 3C) in response to Ag, alone or in mature BMMCs after a 72-h exposure to SCF combination with acute SCF, was reduced in the cSCF-BMMCs To determine whether the switch to a hyporesponsive phenotype compared with BMMCs cultured in IL-3 alone. The production could be induced with reduced exposure times to SCF, newly of the chemokine MCP-1 (CCL2) was also reduced in the cSCF- seeded bone marrow cells were, after initial culturing in media with BMMCs when cells were stimulated with Ag alone, but there was IL-3 alone, exposed to SCF at weekly intervals. Their capacity to little reduction in MCP-1 production when cells were challenged respond to Ag was then examined as they approached maturity with Ag and acute SCF concurrently (Fig. 3D), potentially (4 wk). These experiments revealed that exposure to SCF for as reflecting differential transcriptional regulation. In contrast to little as 1 wk prior to reaching maturity (4 wk) diminished the acute SCF, PGE2 was still capable of interacting synergistically ability of Ag to elicit degranulation (data not shown). To further 5432 SCF AND MAST CELL ACTIVATION PROGRAMMING define the kinetics of this switch in phenotype and to determine mature BMMCs developed in the SCF-containing cultures leads to whether this switch could be elicited in the mature cultures, SCF apoptosis (31), we attempted to analyze reversibility of the phe- was added to 4-wk-old IL-3–generated cultures of BMMCs for notype in BMMCs cultured in the presence of SCF for the initial various periods up to 5 d, and Ag-mediated degranulation was 2 wk and then cultured in IL-3 alone for an additional 2 wk. Under examined. We observed that 48–72 h of exposure to SCF was such conditions, the majority of cells were still viable, as deter- required before there was substantial evidence of downregulation mined by Annexin V staining (data not shown). As shown in Fig. of Ag-mediated degranulation and that maximal attenuation re- 4B, removal of SCF from cSCF-BMMCs allows at least a partial quired .120 h of exposure (Fig. 4A). These data demonstrate that reversion of these cells from the SCF-generated hypoactive the switch in activation phenotype, following introduction of SCF, phenotype. is also observed in mature BMMCs. Therefore, this switch was not a consequence of cryptic alterations during cell development. SCF-mediated downregulation of mediator release is concentration dependent The hyporesponsive phenotype is partly reversible To examine whether the SCF-induced downregulation was “tun- We next determined whether the hyporesponsive phenotype was able,” bone marrow cells were cultured in the presence of IL-3 reversible upon removal of SCF. Because removal of SCF from with increasing concentrations of SCF. As shown in Fig. 4C, the responsiveness to Ag was inversely proportional to increasing concentration of SCF, demonstrating that SCF can produce a A graded MC hyporesponsiveness phenotype dictated by its con-

centration. Downloaded from SCF-mediated downregulation of mediator release minimally affects signaling processes leading to calcium mobilization FcεRI-mediated activation of MCs requires a complex signaling cascade that leads to the activation of phospholipase (PL)Cg

and PI3K, ultimately resulting in an increase in cytosolic calcium http://www.jimmunol.org/ concentrations, and protein kinase C (PKC) activation, which are B obligatory signals for MC activation. In addition, such pathways, in association with those regulated by MAPKs, lead to cytokine production (1, 29, 32, 33). Therefore, we examined critical sig- naling events initiated by FcεRI and SCF in the cSCF-BMMCs, following overnight cytokine deprivation, in an effort to identify the defect that would explain the hypoactive phenotype. We ini- tially examined the of key molecules in the by guest on September 25, 2021 PLCg-activation pathway (LAT, PLCg1, PLCg2, and Btk), PI3K- regulated signaling molecules (AKT and RSK), and MAPKs (ERK1/2, JNK, and p38). As can be seen, we observed very minor (albeit statistically significant) reductions in the phosphorylation C of Btk, PLCg1, PLCg2, LAT, and JNK but no changes in other molecules examined (including AKT, as a surrogate marker for PI3K activation; RSK; ERK; and p38) (Fig. 5A, 5B). With the exception of Btk and PLCg2, this reflected a similar minor re- duction in protein level (Fig. 5C). To examine whether these changes were of sufficient magnitude to influence downstream signaling, we next examined whether the consequential calcium signal or PKC translocation/activation was attenuated in the cells cultured in SCF. As shown in Fig. 6A, culturing of BMMCs in SCF had no impact on the calcium signal in response to Ag in the FIGURE 4. Kinetics of onset, reversibility of SCF-induced hypores- absence or presence of acute SCF. Furthermore, there was no ponsive phenotype, and effect of different SCF concentration in culture. evidence of defects in Ag-mediated PKC translocation in the (A) BMMCs were cultured in IL-3 alone for 4 wk and then SCF was added to the cultures for the periods indicated. After sensitization, the cells were cSCF-BMMCs (Fig. 6B). Taken together, these data demonstrate sensitized overnight with IgE in IL-3–containing culture medium prior to that the hyporesponsive phenotype observed in the cSCF-BMMCs or during culture with SCF, depending on the time point examined. The is not explained by underlying defects in major regulatory path- cells were then stimulated with the indicated concentrations of Ag (DNP- ways responsible for the calcium signal following FcεRI aggrega- HSA) for 30 min, and degranulation (b-hex) was determined. These data tion and, by inference, upstream signals regulating these processes. were compared with the release from BMMCs grown for the entire 6 wk in In addition, the lack of modifications of other major regulatory IL-3 alone (IL-3) or in IL-3 and SCF (IL-3+SCF). (B) Two-week-old processes, including MAPKs and PKC, did not appear to be the BMMCs cultured in IL-3 and SCF were transferred into IL-3 only-con- contributory factors to the profound hyporesponsiveness of the taining media and cultured for an additional 2 wk; degranulation of these cSCF-BMMCs. cells [IL-3+(SCF; removed at second wk)] along with cells grown in IL-3 or IL-3+SCF–containing media for 4 wk was determined. (C) BMMCs Hck is downregulated following prolonged exposure of MCs to were grown in IL-3 in the presence or absence of SCF (0–100 ng/ml) for 4 SCF wk, and degranulation (b-hex) was determined. Data represent mean and SEM [(A, C) n =3;(B) n = 6], and the differences between IL-3+SCF– and In seeking to identify an alternative explanation for the reduced IL-3+(SCF; removed at second wk)-cultured cells are indicated. *p , FcεRI-mediated responses in the cSCF-BMMCs, we explored 0.05, t test. more global changes in expression of signaling proteins by gene- The Journal of Immunology 5433 Downloaded from

FIGURE 6. Extended SCF exposure does not affect the calcium signal or PKC translocation in response to acute challenge with Ag and/or SCF. (A) BMMCs were cultured for 4 wk in IL-3 in the absence or presence of SCF and sensitized overnight in cytokine-free media. Fura 2-loaded cells were then challenged with Ag (DNP-HSA; 10 ng/ml) and/or SCF (10 ng/

ml), as indicated, and calcium response was analyzed. (B) BMMCs were http://www.jimmunol.org/ prepared and sensitized as in (A) and then cells were stimulated with Ag (10 ng/ml) or the positive control (PMA; 500 nM) for 2 min. The mem- brane fractions were isolated and analyzed by immunoblotting. Data in (A) represent mean and SEM (n = 10). In (B), the blots are representative of three mice.

either upregulated or downregulated in the cells grown in IL-3+ SCF compared with the cells grown in IL-3 alone (Supplemental Table I). Some of the more notable, but predictable, differences by guest on September 25, 2021 were the marked increases in mRNA for the MC proteases, tryptase a/b 1 and MC protease 4, which would reflect the in- creased granularity observed in the cSCF-BMMCs (Supplemental Fig. I). Few, if any, of the downregulated by SCF would be predicted to influence FcεRI-mediated degranulation, with the exception of the Src kinase hck (Supplemental Table I, high- lighted), which was one of the most highly downregulated genes in the cSCF-BMMCs. This downregulation of hck was of particular interest because Hck-deficient BMMCs exhibit defective Ag-mediated degranula- tion and cytokine generation without impairment in the calcium signal (34), exactly as noted in this study for the cSCF-BMMCs. To confirm that prolonged exposure to SCF downregulated Hck expression in BMMCs, we assayed for Hck mRNA and protein in the cSCF-treated cells. As shown in Fig. 7A, as observed in the gene-expression array, there was a marked reduction in hck mRNA FIGURE 5. Impact of extended SCF exposure on BMMC signaling in in the cSCF-BMMCs when examined by real-time PCR. Fur- A response to acute challenge with Ag or SCF. ( ) BMMCs were cultured for thermore, there was a pronounced reduction in Hck protein in the 4 wk in IL-3 in the absence or presence of SCF and sensitized overnight in cSCF-BMMCs (Fig. 7B). Downregulation of Hck protein was also cytokine-free media. The cells were then challenged with Ag (DNP-HSA; 10 ng/ml) or SCF (10 ng/ml) for 2 min, lysed, and analyzed by immuno- observed at earlier times of SCF exposure (7 d) (data not shown). blotting. (B) The proteins in (A) were evaluated. (C) Protein expression of To determine whether we could reverse the hypoactive pheno- the significantly regulated signaling molecules analyzed in (B) was evalu- type by ectopic expression of Hck, we attempted to overexpress ated. The blots in (A) are representative of three mice. Data in (B) and (C) Hck in BMMCs cultured in the absence and presence of SCF, as represent mean and SEM (n = 5 and n = 3, respectively). *p , 0.05, t test. described (34). As shown in Fig. 7C, although overexpression of Hck was achieved in the BMMCs cultured in the absence of SCF, no such overexpression was observed in the cells cultured in SCF. expression microarray. BMMCs were developed from six litter- Thus, we were unable to determine whether ectopic expression of mate male mice and cultured in IL-3 in the absence or presence of Hck would reverse the hyporesponsive phenotype in the cSCF- SCF for 4 wk, mRNA was extracted, and then relative gene ex- BMMCs. Nevertheless, these results support our conclusion that pression was examined. As expected, a wide array of genes was SCF reduces Hck expression at the mRNA level. 5434 SCF AND MAST CELL ACTIVATION PROGRAMMING

cSCF-BMMCs. Furthermore, immunofluorescence microscopy re- vealed that there was also a marked decrease in a-tubulin poly- merization in the cSCF-BMMCs (Fig. 8D, 8E). Taken together, these data are consistent with the conclusion that the SCF-induced hyporesponsive phenotype is a consequence of ineffective cyto- skeletal reorganization required for mediator release, potentially as a consequence of downregulation of the Hck expression or other signaling proteins downstream of calcium mobilization.

Discussion The results of this study demonstrate that MCs chronically exposed to SCF have a significantly reduced capacity to degranulate and to generate cytokines in response to Ag. This phenotype was linked to ineffective cytoskeletal reorganization potentially associated with downregulation of Hck. Thus, these findings provide evidence for a novel mechanism by which the extent of MC activation may be programmed through the influence of cytokines present within the immediate microenvironment. Based on the relative lack of ability of long-term SCF exposure Downloaded from to reduce the capacity of PMA/ionomycin, adenosine, and PGE2 to enhance Ag-mediated degranulation and cytokine production, the hyporesponsive phenotype induced by SCF appears to be re- stricted to MC responses mediated by receptors that signal through tyrosine kinases rather than G proteins. Together with the ex-

tended time period (.72 h) required for the onset of inhibition by http://www.jimmunol.org/ SCF and the lack of effects on FcεRI and KIT expression, we conclude that SCF induced the hyporesponsive phenotype through changes in the expression of specific critical signaling protein(s) rather than as a consequence of short-term posttranslational modification (e.g., through protein phosphorylation). The lack of marked defects in inducible critical early and intermediary sig- naling events, including regulators of calcium mobilization, PI3K, FIGURE 7. Extended SCF exposure downregulates mRNA and protein PKC, and MAPKs, in the hyporesponsive phenotype, further expression of Hck in BMMCs. (A) BMMCs were cultured for 4 wk in IL-3 demonstrated that such downregulation was a consequence of a by guest on September 25, 2021 in the absence or presence of SCF, and expression of Hck mRNA was restricted targeting and not merely a generalized phenomenon. B A determined by quantitative real-time PCR. ( ) The cells in ( ) were lysed, The lack of any marked defects in these early signaling events and Hck expression was analyzed by immunoblotting and evaluated. (C) additionally indicated that the activation phenotype observed in the BMMCs from two mice were cultured in IL-3 in the absence or presence of SCF, transfected with retroviral vector pMX-puro with (HCK) or without cSCF-BMMCs likely reflected downregulation of gene products (Ctrl) the mouse hck cDNA, and Hck mRNA expression was determined as that control critical downstream processes. Although the expres- in (A). Data in (A) and (B) represent mean and SEM (n = 3 and n =4, sion array revealed that a multitude of genes is upregulated or respectively), and the differences between IL-3– and IL-3+SCF–cultured downregulated in the cells grown in SCF and IL-3 compared with cells are indicated. *p , 0.05, t test. the cells grown in IL-3 alone, based on the current state of knowledge of MC-signaling processes (2, 29), the vast majority of Thus, although downregulation of hck correlates with, and may the identified genes appeared to have little relevance to the reg- help to explain, the observed change to a hyporesponsive pheno- ulation of MC degranulation and/or cytokine production and re- type, it is also possible that modification of other genes or re- lease. Nevertheless, a notable exception was the substantial re- duction in the levels of critical signaling proteins following duction in the expression of the Src kinase family member hck. proteolytic digestion may contribute to the cSCF-BMMC hypo- Indeed, real-time PCR and Western blot analysis confirmed a responsiveness. marked attenuation of the expression of Hck mRNA and protein in the cSCF-BMMCs. The hypoactive phenotype is associated with defective By using hck2/2 mice and MCs derived from the bone marrow cytoskeletal reorganization of these mice, it was shown that Hck deficiency results in an MC Both the calcium signal and Hck regulate cytoskeletal reorgani- phenotype characterized by defective Ag-mediated degranulation zation through processes that regulate actin polymerization/ and reduced expression of TNF-a and IL-6 (34). The corollary is depolymerization, as well as formation of through that upregulation of Hck activity was shown to produce a hyper- tubulin polymerization. Both of these events are essential for MC responsive MC phenotype (41). Of particular note was the ob- mediator release (35–40). Thus, we examined whether an SCF- servation that defective degranulation and cytokine production in dependent reduction in cytoskeletal reorganization would provide the hck2/2 BMMCs were not associated with a concomitant de- a mechanistic explanation for the attenuated mediator release crease in calcium flux (34), exactly as observed in the cSCF- observed in the cSCF-BMMCs. As revealed by flow cytometry BMMCs. However, it should be noted that not all observations (Fig. 8A) and confirmed by immunofluorescence microscopy (Fig. in the cSCF-BMMCs recapitulated those observed in the hck2/2 8B,8C),FcεRI-mediated actin depolymerization, which is re- BMMCs. In the hck2/2 BMMCs, for example, there was an in- quired for degranulation, was substantially attenuated in the crease in the phosphorylation of Lyn substrates, such as Dok2, as The Journal of Immunology 5435

a consequence of increased Lyn activity. However, we observed that the phosphorylation of Dok2 was slightly reduced in the cSCF-BMMCs (data not shown), suggesting that Lyn activity was not unregulated in these cells. Hck binds to and/or tyrosine phosphorylates a number of ligands/ substrates including WASP, WIP, ELMO1, and a-tubulin (42); with regard to Hck, both WASP (43) and WIP (35) were dem- onstrated to be essential for MC function. Hck, WASP, and WIP in MCs and other cell types (44, 45) are known to regulate cy- toskeletal and/or reorganization (34, 46, 47) through the control of actin and tubulin polymerization/depolymerization, respectively. Certainly, both of these processes are recognized to be essential for MC degranulation (35–40) and, indeed, it is be- lieved that Hck, WASP, and WIP may exert their influence on MC activation through the regulation of such processes. Thus, our findings that Ag-induced actin polymerization was attenuated in the cSCF-BMMCs support the conclusion that the downregulation of Hck and subsequent defective cytoskeletal reorganization may

account for the attenuated degranulation in the cSCF-BMMCs. Downloaded from Nevertheless, it is possible that downregulation of other signal- ing molecules contributes to this phenotype. The mode by which cytoskeletal reorganization would prevent FcεRI/KIT-mediated cytokine production is uncertain; however, because of the reduced total cytokine protein levels observed,

this is unlikely to be solely due to defective trafficking to the cell http://www.jimmunol.org/ surface. A potential explanation is inadequate access of signaling molecules to the nucleus in the cSCF-BMMCs. The process by which SCF leads to downregulation of Hck expression in MCs is also unknown. Thus, both of these issues warrant further in- vestigation. The identification of the MC phenotypic change induced by chronic exposure to SCF led us to conclude that the cytokine milieu to which MCs are exposed in vivo may exert not only homeostatic control of growth and survival but also of activation. The impor- by guest on September 25, 2021 tance of such a mechanism for regulating MC function in a physiological setting may have evolved to temper inappropriate MC activation during SCF-mediated cell division, expansion, and development where the influence of SCF on MCs would be ex- pected to be greatest. In addition, the plasticity of the phenotype in developing cells would allow reversal of the hyporesponsive phenotype to a more normal responsive phenotype when MCs are mature and resident in tissues. Of potential relevance to this ar- gument was our observation that the degree of the phenotypic hyporesponsiveness correlated with the concentration of SCF to which the cells were exposed. Local concentrations of SCF also dictate the described phenotypic differences associated with serosal (connective tissue) compared with mucosal MCs. Thus, it is also possible that SCF similarly dictate the activation capacity of MCs, as a function of their compartmentalization within their resident tissues. Of interest is the report that connective tissue and mucosal MCs display differential gene expression that may impact Ag- mediated degranulation (48). Nevertheless, the gene profiles as- sociated with these phenotypes appear to be different from those observed in our study. For example, although there was markedly reduced expression of FcgRIIB (Fcgr2b), Rab-GEF1 (Rabgef1), FIGURE 8. The effect of extended SCF exposure on actin and a-tubulin and CD81 (Cd81) reported in connective tissue MCs associated polymerization. (A) BMMCs cultured in IL-3 in the absence or presence of SCF were sensitized and then activated with Ag (10 ng/ml) for 0, 2, 5, 10, or 30 min. The cells were fixed/permeabilized, and the amount of F-actin was determined by phalloidin-FITC staining and flow cytometry. (B) The was labeled with anti–a-tubulin/secondary–Alexa Fluor 568 Ab (), and cells in (A) were activated with Ag (10 ng/ml) for 0, 2, or 10 min and fixed/ the nuclei were labeled with DAPI (blue). (E) The fluorescence intensity of permeabilized, F-actin was labeled with phalloidin-FITC (green), and the the visualized cells in (D) was evaluated. Images in (B) and (D) are rep- nuclei were labeled with DAPI (blue). (C) The fluorescence intensity of the resentative of four mice. Data in (A), (C), and (E) represent mean and SEM visualized cells in (B) was evaluated. (D) The cells in (A) were activated (n = 4), and differences between IL-3– and IL-3+SCF–cultured cells are with Ag (10 ng/ml) for 0, 2, or 10 min and fixed/permeabilized, a-tubulin indicated. *p , 0.05, t test. 5436 SCF AND MAST CELL ACTIVATION PROGRAMMING with reduced Ag-mediated degranulation (48), we observed little 9. Lukacs, N. W., S. L. Kunkel, R. M. Strieter, H. L. Evanoff, R. G. Kunkel, M. L. Key, and D. D. Taub. 1996. The role of stem cell factor (c-kit ligand) and change in the expression of these genes in the cSCF-BMMCS inflammatory cytokines in pulmonary mast cell activation. Blood 87: 2262–2268. (Supplemental Table I). 10. Tsai, M., T. Takeishi, H. Thompson, K. E. Langley, K. M. Zsebo, D. D. Metcalfe, Thus, it is of interest whether SCF is unique in its ability to E. N. Geissler, and S. J. Galli. 1991. Induction of mast cell proliferation, mat- ε uration, and heparin synthesis by the c-kit ligand, stem cell factor. Proc. Natl. downregulate Fc RI-mediated MC activation in the manner re- Acad. Sci. USA 88: 6382–6386. ported in this study. It was reported, for example, that IL-10 (49) 11. Hill, P. B., A. J. MacDonald, E. M. Thornton, G. F. Newlands, S. J. Galli, and and TGF-b (50) downregulate MC activation in response to Ag. H. R. Miller. 1996. Stem cell factor enhances immunoglobulin E-dependent mediator release from cultured rat bone marrow-derived mast cells: activation Although, the mechanism for the downregulation by TGF-b is of previously unresponsive cells demonstrated by a novel ELISPOT assay. Im- unknown (50) or may be due to reduction in cell survival (51), the munology 87: 326–333. 12. Hundley, T. R., A. M. Gilfillan, C. Tkaczyk, M. V. Andrade, D. D. Metcalfe, and IL-10–mediated downregulation could be explained by downreg- M. A. Beaven. 2004. Kit and FcεRI mediate unique and convergent signals for ulation of FcεRI expression (49). However, this is not the case release of inflammatory mediators from human mast cells. Blood 104: 2410–2417. with the hyporesponsive phenotype induced by SCF reported in 13. Tkaczyk, C., V. Horejsi, S. Iwaki, P. Draber, L. E. Samelson, A. B. Satterthwaite, D. H. Nahm, D. D. Metcalfe, and A. M. Gilfillan. 2004. NTAL phosphorylation this article. Given the role of MCs in allergic and autoimmune is a pivotal link between the signaling cascades leading to human mast cell disorders (52), if these agents or the pathways that they regulate degranulation following Kit activation and FcεRI aggregation. Blood 104: 207– could be used to downregulate MC activation in a clinical setting, 214. 14. Wershil, B. K., M. Tsai, E. N. Geissler, K. M. Zsebo, and S. J. Galli. 1992. 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Bloes, and could result in exaggerated mediator release, with the potential of R. L. Stevens. 1985. Fibroblasts maintain the phenotype and viability of the rat heparin-containing mast cell in vitro. J. Immunol. 135: 3454–3462. leading to MC-driven pathology. Such observations may aid in the 19. Choi, O. H., J. H. Lee, T. Kassessinoff, J. R. Cunha-Melo, S. V. P. Jones, and design of novel approaches to downregulate MC activation, pro- M. A. Beaven. 1993. Antigen and carbachol mobilize calcium by similar viding potential therapeutic approaches for the treatment of al- mechanisms in a transfected mast cell line (RBL-2H3 cells) that expresses ml muscarinic receptors. J. Immunol. 151: 5586–5595. lergic disorders. 20. Tkaczyk, C., D. D. Metcalfe, and A. M. Gilfillan. 2002. Determination of protein phosphorylation in FcεRI-activated human mast cells by immunoblot analysis

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Supplemental figure legends

Table S1. Gene chip analysis.

Relative gene expression in BMMCs cultured for 4 weeks in the IL-3 (30 ng/ml) and IL-3+SCF (100 ng/ml). The cells were processed and arrays conducted as described in supplemental methods. The data were generated from BMMCs cultured from 6 littermate male mice and the values represent the mean fold differences in relative expression.

Figure S1. Effects of SCF on cell growth, receptor expression and morphology.

(A) Growth of BMMCs cultured in IL-3 in the absence or presence of SCF. Bone marrow cells were cultured in media containing IL-3 (30 ng/ml) or IL-3+SCF (100 ng/ml), cell density was kept up to 1×106 cells/ml, and the cells were counted once a week for 8 weeks. The inserted graph represents different scale for IL-3 only-cultured BMMCs. (B-C) Four week old BMMCs, cultured in the absence (B) or presence (C) of SCF as above were stained with FITC-labeled anti-FcεRI α- chain and PE-labeled anti-KIT and cell labeling was determined by flow cytometry. (D-E) Cytospins of BMMCs cultured as above were stained with

Toluidine blue (D) or Alcian blue/ Safranin (E). In A, data represent means and

SEM (n=4). In B and E, the images are representative of 3 experiments conducted on separate cell preparations.

2

Figure S2. Effects of IL-3 culturing without or with SCF on granule content after activation with antigen.

BMMCs were cultured in IL-3 in the absence or presence of SCF and IgE sensitized cells were (Ag, DNP-HSA; 10 ng/ml), or not (Control) activated by antigen for 30 min at 37 C. Cytospins of the cells were stained with Toluidine blue and analyzed. The images are representative of 3 experiments conducted on separate cell preparations.

Figure S3. Morphology of BMMCs co-cultured with NIH 3T3 fibroblasts and limited retention of IL-6 in BMMCs following antigen activation.

BMMCs co-cultured with NIH 3T3 cells as described in methods were stained with Toluidine blue (A) or Alcian blue/ Safranin (B) and the cytospins analyzed.

(C) BMMCs were cultured in IL-3 in the absence (solid bars) or presence (white bars) of SCF for 4 weeks as indicated. The IgE sensitized cells in cytokine-free media were then challenged for 6 h with antigen (Ag, DNP-HSA) in the presence or absence of SCF, and amount of IL-6 released into the media was determined

(Released cytokines). Following activation, the cells were lysed and IL-6 in the the remaining cellular contents was determined (Retained cytokines; note the different scales on the Y axes). In A and B, the images are representative of 3 experiments conducted on separate cell preparations. In C and D, the data represent means and SEM (n=3) and differences between IL-3 and IL-3+SCF- cultured cells are indicated (*, p <0.05, t test). Gene Test= IL3+SCF signal FDR corrected Probe Set ID Gene Title Symbol / Control=IL3+ signal p-value DOWN 1418095_at small muscle protein, X-linked Smpx -185.88 6.30E-12 1420583_a_at RAR-related alpha Rora -39.60 1.57E-11 1425603_at transmembrane protein 176A Tmem176a -34.12 3.74E-10 1455165_at RAR-related orphan receptor alpha Rora -32.84 7.28E-14 1436325_at RAR-related orphan receptor alpha Rora -29.24 8.49E-11 1418004_a_at transmembrane protein 176B Tmem176b -29.04 5.40E-10 1423909_at transmembrane protein 176A Tmem176a -26.58 5.87E-09 1416236_a_at protein zero-like 2 Mpzl2 -23.51 4.63E-04 1421471_at Y receptor Y1 Npy1r -22.60 4.61E-04 1449455_at hemopoietic cell kinase Hck -21.98 4.48E-09 1449175_at G-protein coupled receptor 65 Gpr65 -20.79 1.15E-07 1424034_at RAR-related orphan receptor alpha Rora -20.71 1.15E-12 1418782_at gamma Rxrg -18.54 4.05E-06 1458129_at RAR-related orphan receptor alpha Rora -17.96 5.64E-11 1441811_x_at transmembrane protein 176A Tmem176a -17.30 1.49E-09 1450020_at chemokine (C-X3-C) receptor 1 Cx3cr1 -15.72 5.22E-09 1436326_at RAR-related orphan receptor alpha Rora -15.71 6.42E-09 1449220_at GTPase, IMAP family member 3 /// sGimap3 /// -15.47 8.97E-10 1448499_a_at epoxide 2, cytoplasmic Ephx2 -13.43 2.66E-04 1460602_at deleted in cancer 1 Dlc1 -12.86 1.63E-08 1422134_at FBJ osteosarcoma oncogene B Fosb -11.96 2.51E-04 1416022_at fatty acid binding protein 5, epidermaFabp5 -10.98 8.47E-08 1456250_x_at transforming growth factor, beta induTgfbi -10.74 7.29E-07 1424111_at -like growth factor 2 receptor Igf2r -10.56 5.51E-08 1442542_at eyes absent 4 homolog (Drosophila) Eya4 -10.28 3.85E-09 1452757_s_at hemoglobin alpha, adult chain 1 /// heHba-a1 /// H -10.04 6.14E-10 1448123_s_at transforming growth factor, beta induTgfbi -10.00 6.12E-07 1427345_a_at sulfotransferase family 1A, phenol-prSult1a1 -9.94 5.74E-07 1451634_at antisense Igf2r RNA Airn -9.89 2.84E-07 1436173_at deleted in liver cancer 1 Dlc1 -9.76 2.23E-09 1436823_x_at hemoglobin Y, beta-like embryonic c Hbb-y -9.43 8.84E-08 1435981_at navigator 2 Nav2 -9.41 1.61E-07 1436717_x_at hemoglobin Y, beta-like embryonic c Hbb-y -9.02 6.54E-09 1451204_at scavenger receptor class A, member 5Scara5 -9.00 3.77E-10 1447500_at cut-like 2 Cux2 -8.96 1.16E-07 1422009_at ATPase, Na+/K+ transporting, beta 2Atp1b2 -8.94 2.03E-04 1415871_at transforming growth factor, beta induTgfbi -8.83 1.03E-04 1435148_at ATPase, Na+/K+ transporting, beta 2Atp1b2 -8.36 1.28E-10 1422245_a_at MRV integration site 1 Mrvi1 -8.08 5.79E-05 1440801_s_at kinase, beta 2 Adrbk2 -8.02 1.71E-06 1450019_at chemokine (C-X3-C) receptor 1 Cx3cr1 -7.99 2.85E-08 1454984_at leukemia inhibitory factor receptor Lifr -7.90 4.21E-07 1457108_at ------7.83 1.44E-05 1425528_at paired related homeobox 1 Prrx1 -7.80 1.27E-08 1418804_at succinate receptor 1 Sucnr1 -7.73 2.72E-08 1437463_x_at transforming growth factor, beta induTgfbi -7.70 2.69E-08 1455101_at and actin regulator 2 Phactr2 -7.64 7.60E-08 1437498_at predicted gene 9971 Gm9971 -7.38 2.80E-05 1450621_a_at hemoglobin Y, beta-like embryonic c Hbb-y -7.35 7.43E-09 1428774_at 6 Gpc6 -7.11 8.96E-06 1451612_at metallothionein 1 Mt1 -7.07 1.23E-07 1455869_at ------7.04 8.18E-07 1460480_at endogenous retroviral sequence 3 Erv3 -7.02 7.72E-10 1420544_at germinal center expressed transcript 2Gcet2 -6.98 7.23E-10 1423231_at neurogranin Nrgn -6.95 2.99E-09 1447980_s_at small G protein signaling modulator 1Sgsm1 -6.95 2.19E-05 1416505_at subfamily 4, group ANr4a1 -6.90 8.00E-04 1428361_x_at hemoglobin alpha, adult chain 1 /// heHba-a1 /// H -6.75 1.52E-04 1429206_at Rho-related BTB domain containing Rhobtb1 -6.69 2.35E-06 1432959_at RIKEN cDNA 4930447I22 gene 4930447I22 -6.61 1.26E-05 1431182_at heat shock protein 8 /// hypothetical LHspa8 /// L -6.54 2.04E-05 1415949_at carboxypeptidase E /// similar to carbCpe /// LOC -6.53 1.42E-08 1452404_at phosphatase and actin regulator 2 Phactr2 -6.37 3.79E-10 1448785_at runt-related 1; tra Runx1t1 -6.35 1.41E-09 1447046_at Uncoupling protein 2 (mitochondrial,Ucp2 -6.29 8.32E-06 1421571_a_at similar to Lymphocyte antigen 6C preLOC10004 -6.28 3.73E-10 1444566_at Uncoupling protein 2 (mitochondrial,Ucp2 -6.26 2.47E-06 1443969_at substrate 2 Irs2 -6.25 2.09E-07 1425815_a_at hyaluronan mediated motility receptoHmmr -6.25 2.53E-08 1459665_s_at MRV integration site 1 Mrvi1 -6.24 2.05E-05 1416021_a_at fatty acid binding protein 5, epidermaFabp5 /// G -6.15 1.86E-08 1456446_at RIKEN cDNA 4930523C07 gene 4930523C0 -6.13 4.86E-09 1435172_at homolog (Xenopus laeEomes -6.06 1.45E-08 1440200_at family with sequence similarity 184, Fam184b -6.03 2.39E-11 1452291_at ArfGAP with RhoGAP domain, ankyArap2 -6.01 8.76E-08 1427161_at centromere protein F Cenpf -5.93 1.06E-07 1418981_at caspase 12 Casp12 -5.92 6.09E-04 1422189_x_at T-cell receptor gamma, variable 4 Tcrg-V4 -5.89 7.01E-09 1442582_at germinal center expressed transcript 2Gcet2 -5.80 8.06E-11 1450521_a_at T-cell receptor gamma, variable 4 Tcrg-V4 -5.79 4.87E-09 1430811_a_at NUF2, NDC80 kinetochore complex Nuf2 -5.71 8.30E-09 1450611_at 3 Orm3 -5.71 5.50E-04 1453226_at family member 18B Kif18b -5.68 5.65E-04 1426001_at eomesodermin homolog (Xenopus laeEomes -5.63 4.79E-10 1452519_a_at protein 36 Zfp36 -5.62 2.75E-04 1425526_a_at paired related homeobox 1 Prrx1 -5.60 1.12E-06 1432960_at RIKEN cDNA 4930447I22 gene 4930447I22 -5.59 1.86E-05 1439568_at gene regulated by in breast cGreb1 /// L -5.58 8.98E-06 1438061_at RIKEN cDNA 4930523C07 gene 4930523C0 -5.49 2.15E-08 1417701_at 1, regulatory (inhPpp1r14c -5.44 1.64E-07 1442878_at peroxiredoxin 6 Prdx6 -5.42 6.80E-09 1449297_at caspase 12 Casp12 -5.41 7.38E-06 1424278_a_at baculoviral IAP repeat-containing 5 Birc5 -5.39 1.99E-06 1453590_at ADP-ribosylation factor-like 5B Arl5b -5.39 8.24E-06 1424112_at insulin-like growth factor 2 receptor Igf2r -5.35 2.02E-04 1421964_at Notch gene homolog 3 (Drosophila) Notch3 -5.29 9.53E-06 1439774_at paired related homeobox 1 Prrx1 -5.28 5.82E-06 1431213_a_at predicted gene 3579 Gm3579 -5.25 2.11E-07 1422188_s_at T-cell receptor gamma, variable 2 /// Tcrg-V2 /// -5.24 1.64E-08 1451355_at alkaline 2 Acer2 -5.18 2.26E-06 1419152_at RIKEN cDNA 2810417H13 gene 2810417H1 -5.17 2.07E-07 1433741_at CD38 antigen Cd38 -5.12 5.77E-05 1448046_at Rab9 effector protein with kelch motiRabepk -5.05 7.20E-07 1456077_x_at cell division cycle 25 homolog C (S. Cdc25c -4.95 1.18E-04 1418736_at UDP-GalNAc:betaGlcNAc beta 1,3-gB3galnt1 -4.93 4.97E-04 1434423_at GULP, engulfment adaptor PTB domGulp1 -4.88 2.70E-08 1434091_at fatty acid amide hydrolase Faah -4.88 1.29E-04 1452040_a_at cell division cycle associated 3 Cdca3 -4.88 5.58E-07 1433600_at adrenergic receptor, alpha 2a Adra2a -4.83 1.61E-06 1416299_at Shc SH2-domain binding protein 1 Shcbp1 -4.76 3.43E-10 1456634_at RIKEN cDNA 9830001H06 gene 9830001H0 -4.74 1.01E-03 1416258_at thymidine kinase 1 Tk1 -4.71 9.38E-09 1438934_x_at sema domain, immunoglobulin doma Sema4a -4.66 3.20E-06 1439736_at RIKEN cDNA 5830453J16 gene 5830453J1 -4.62 6.86E-07 1417910_at cyclin A2 Ccna2 -4.62 7.55E-08 1447363_s_at budding uninhibited by benzimidazol Bub1b -4.62 1.27E-07 1452242_at centrosomal protein 55 Cep55 -4.59 2.70E-06 1437187_at transcription factor 7 E2f7 -4.57 6.53E-05 1450758_at contactin associated protein-like 2 Cntnap2 -4.55 5.87E-08 1441015_at ------4.53 3.25E-06 1448899_s_at RAD51 associated protein 1 Rad51ap1 -4.50 1.14E-07 1447979_at small G protein signaling modulator 1Sgsm1 -4.45 2.18E-04 1448925_at twist homolog 2 (Drosophila) Twist2 -4.44 3.00E-09 1421965_s_at Notch gene homolog 3 (Drosophila) Notch3 -4.44 1.06E-06 1441286_at predicted gene 13948 Gm13948 -4.42 5.03E-05 1415899_at Jun-B oncogene Junb -4.41 3.47E-05 1438210_at G protein-coupled receptor 149 Gpr149 -4.38 5.04E-06 1442881_at ------4.35 8.15E-04 1448830_at dual specificity phosphatase 1 Dusp1 -4.35 8.12E-06 1439874_at RIKEN cDNA 9330102E08 gene 9330102E0 -4.34 1.21E-05 1415904_at lipoprotein Lpl -4.33 3.74E-09 1439040_at centromere protein E Cenpe -4.28 1.73E-07 1416756_at DnaJ (Hsp40) homolog, subfamily B,Dnajb1 -4.24 5.37E-05 1456280_at claspin homolog (Xenopus laevis) Clspn -4.24 9.95E-07 1452004_at /calcitonin-related polypeptCalca -4.23 9.73E-05 1432129_a_at paired related homeobox 1 Prrx1 -4.20 2.31E-06 1425452_s_at family with sequence similarity 84, mFam84a -4.15 9.68E-07 1452958_at aspartate beta-hydroxylase domain coAsphd2 -4.14 2.06E-05 1448575_at interleukin 7 receptor Il7r -4.09 1.56E-04 1439377_x_at cell division cycle 20 homolog (S. cerCdc20 -4.08 9.59E-07 1430574_at cyclin-dependent kinase inhibitor 3 Cdkn3 -4.04 2.32E-07 1453557_at RIKEN cDNA 4930562F07 gene 4930562F0 -4.03 4.69E-08 1424046_at budding uninhibited by benzimidazol Bub1 -4.03 9.01E-08 1419088_at tissue inhibitor of metalloproteinase 3Timp3 -4.01 2.51E-07 1448110_at sema domain, immunoglobulin doma Sema4a -4.01 9.77E-06 1417409_at Jun oncogene Jun -3.98 2.12E-04 1456636_at RIKEN cDNA 4930414L22 gene 4930414L2 -3.94 7.22E-05 1419091_a_at annexin A2 Anxa2 -3.94 2.84E-05 1434437_x_at ribonucleotide reductase M2 Rrm2 -3.92 3.47E-05 1438763_at , axonemal, heavy chain 2 Dnahc2 -3.86 1.52E-05 1437611_x_at kinesin family member 2C Kif2c -3.86 8.82E-09 1434450_s_at adrenergic receptor kinase, beta 2 Adrbk2 -3.85 1.79E-09 1417938_at RAD51 associated protein 1 Rad51ap1 -3.84 7.35E-10 1418106_at hairy/enhancer-of-split related with YHey2 -3.83 2.21E-06 1419161_a_at NADPH oxidase 4 Nox4 -3.82 3.99E-08 1435845_at ------3.82 4.17E-07 1434496_at polo-like kinase 3 (Drosophila) Plk3 -3.82 2.31E-05 1429350_at EP300 interacting inhibitor of differe Eid3 -3.80 4.52E-05 1449335_at tissue inhibitor of metalloproteinase 3Timp3 -3.80 1.14E-08 1415810_at ubiquitin-like, containing PHD and RUhrf1 -3.78 4.71E-06 1416076_at cyclin B1 /// predicted gene 5593 /// pCcnb1 /// G -3.78 2.16E-04 1430581_at BCL2-associated transcription factor Bclaf1 -3.77 3.20E-07 1441115_at DNA segment, Chr 18, ERATO Doi 2D18Ertd23 -3.76 4.02E-09 1431623_at RIKEN cDNA 1700023H06 gene 1700023H0 -3.76 1.06E-04 1456665_at eyes absent 4 homolog (Drosophila) Eya4 -3.75 5.15E-06 1416630_at inhibitor of DNA binding 3 Id3 -3.74 1.52E-07 1418901_at CCAAT/enhancer binding protein (C Cebpb -3.72 7.04E-06 1452314_at Kif11 -3.71 4.94E-06 1416309_at nucleolar and spindle associated prot Nusap1 -3.66 3.56E-06 1450920_at cyclin B2 Ccnb2 -3.66 1.19E-07 1450790_at Tg -3.66 5.87E-08 1456311_x_at establishment of cohesion 1 homolog Esco2 -3.66 5.15E-08 1441284_at ------3.65 8.72E-07 1440109_at DNA segment, Chr 7, ERATO Doi 4 D7Ertd413 -3.65 3.67E-04 1460319_at fucosyltransferase 8 Fut8 -3.65 4.33E-10 1422798_at contactin associated protein-like 2 Cntnap2 -3.64 3.02E-08 1427329_a_at immunoglobulin heavy chain 6 (heav Igh-6 -3.63 6.30E-04 1445467_at ------3.63 4.54E-08 1443604_at ------3.61 5.18E-07 1448929_at coagulation factor XIII, A1 subunit F13a1 -3.60 2.29E-06 1439750_at ------3.59 4.99E-08 1425624_at EPM2A (laforin) interacting protein Epm2aip1 -3.59 1.49E-05 1419297_at histocompatibility 2, O region alpha lH2-Oa -3.57 1.16E-09 1429171_a_at non-SMC condensin I complex, subu Ncapg -3.56 8.59E-06 1460368_at , palmitoylated 4 (MMpp4 -3.56 7.22E-06 1427119_at peptidase inhibitor, Kazal typeSpink4 -3.56 8.20E-08 1441163_at mediator of RNA polymerase II trans Med12l -3.55 1.83E-09 1439153_at ring finger protein 144B Rnf144b -3.53 1.69E-04 1459021_at ------3.52 1.21E-05 1427222_a_at seminal vesicle secretory protein 4 Svs4 -3.51 1.83E-04 1449334_at tissue inhibitor of metalloproteinase 3Timp3 -3.51 3.86E-08 1419816_s_at ERBB receptor feedback inhibitor 1 Errfi1 -3.50 6.67E-05 1450544_at coiled-coil domain containing 48 Ccdc48 -3.50 7.10E-05 1435575_at kinetochore associated 1 Kntc1 -3.50 1.38E-06 1419082_at serine (or cysteine) peptidase inhibitoSerpinb2 -3.49 1.53E-06 1437488_at solute carrier family 9 (sodium/hydroSlc9a9 -3.49 2.46E-07 1416125_at FK506 binding protein 5 Fkbp5 -3.48 1.94E-06 1436723_at centromere protein I Cenpi -3.48 9.51E-05 1427707_a_at Scl/Tal1 interrupting Stil -3.47 7.63E-04 1456367_at fucosyltransferase 8 Fut8 -3.46 1.74E-08 1438307_at high mobility group box 2 Hmgb2 -3.46 1.18E-05 1455695_at ST8 alpha-N-acetyl-neuraminide alphSt8sia1 -3.45 2.42E-08 1442280_at DNA segment, Chr 2, ERATO Doi 75D2Ertd750 -3.44 4.43E-04 1451069_at proviral integration site 3 Pim3 -3.44 3.24E-06 1437900_at RIKEN cDNA 4930523C07 gene 4930523C0 -3.43 8.03E-08 1449851_at period homolog 1 (Drosophila) Per1 -3.43 3.99E-06 1440663_at ------3.43 8.61E-07 1452954_at ubiquitin-conjugating E2C Ube2c -3.43 1.40E-04 1425919_at NADH dehydrogenase (ubiquinone) Ndufa12 -3.42 2.82E-04 1455166_at ADP-ribosylation factor-like 5B Arl5b -3.42 1.31E-05 1449382_at solute carrier family 6 (neurotransmitSlc6a12 -3.42 5.08E-09 1437884_at ADP-ribosylation factor-like 5B Arl5b -3.41 4.60E-05 1454694_a_at topoisomerase (DNA) II alpha Top2a -3.38 6.76E-09 1443027_at ------3.36 3.11E-09 1453103_at actin-binding LIM protein 1 Ablim1 -3.34 5.04E-08 1437181_at pellino 2 Peli2 -3.34 2.91E-08 1460218_at CD52 antigen Cd52 -3.32 5.68E-07 1446553_at ------3.32 4.70E-08 1423521_at B1 Lmnb1 -3.30 4.15E-07 1459962_at RIKEN cDNA 4930523C07 gene 4930523C0 -3.29 2.34E-07 1428393_at neuritin 1 Nrn1 -3.25 1.85E-08 1455609_at citron Cit -3.24 1.63E-04 1452305_s_at centromere protein N Cenpn -3.24 9.06E-04 1448113_at 1 Stmn1 -3.23 1.03E-07 1435306_a_at kinesin family member 11 Kif11 -3.23 7.04E-05 1417828_at 8 Aqp8 -3.23 7.57E-08 1448226_at ribonucleotide reductase M2 Rrm2 -3.23 2.34E-06 1437605_at nephrosis 2 homolog, podocin (huma Nphs2 -3.22 1.47E-04 1418936_at v- musculoaponeurotic fibrosarcoMaff -3.21 5.50E-05 1455257_at beta 3 Itgb3 -3.21 1.04E-05 1448314_at cyclin-dependent kinase 1 Cdk1 -3.21 1.69E-06 1420685_at GRB2-related adaptor protein 2 /// simGrap2 /// L -3.20 8.64E-07 1419089_at tissue inhibitor of metalloproteinase 3Timp3 -3.20 3.84E-07 1433719_at solute carrier family 9 (sodium/hydroSlc9a9 -3.20 3.29E-08 1452073_at family with sequence similarity 64, mFam64a -3.19 1.01E-04 1456139_at antisense Igf2r RNA Airn -3.19 4.15E-04 1448928_at histone deacetylase 6 Hdac6 -3.17 3.45E-07 1433893_s_at sperm associated antigen 5 Spag5 -3.16 8.29E-08 1417821_at DNA segment, Chr 17, human D6S56D17H6S56 -3.15 7.42E-05 1417911_at cyclin A2 Ccna2 -3.15 1.61E-09 1431087_at SPC24, NDC80 kinetochore complexSpc24 -3.15 6.00E-06 1424060_at nei like 3 (E. coli) Neil3 -3.15 5.03E-07 1439997_at 1 Synj1 -3.13 4.09E-05 1426817_at antigen identified by monoclonal antiMki67 -3.13 5.73E-07 1433543_at anillin, actin binding protein Anln -3.12 4.63E-08 1416271_at PERP, TP53 apoptosis effector Perp -3.12 1.06E-04 1449903_at cytotoxic and regulatory T cell molecCrtam -3.12 3.04E-05 1432520_at immunoglobulin heavy chain (J558 faIgh-VJ558 -3.10 2.09E-04 1419386_at 13, epithelial transmembrane Muc13 -3.10 1.41E-05 1435511_at synapsin II Syn2 -3.10 3.67E-07 1437449_at radical S-adenosyl domai Rsad1 -3.10 1.72E-05 1448823_at chemokine (C-X-C motif) ligand 12 Cxcl12 -3.09 7.44E-06 1448627_s_at PDZ binding kinase Pbk -3.08 9.45E-07 1448231_at FK506 binding protein 5 Fkbp5 -3.08 5.76E-07 1438009_at predicted gene 11276 /// histone clustGm11276 / -3.08 8.51E-06 1453771_at GULP, engulfment adaptor PTB domGulp1 -3.07 1.02E-06 1418281_at RAD51 homolog (S. cerevisiae) Rad51 -3.07 7.20E-04 1452703_at S-adenosylhomocysteine hydrolase-liAhcyl2 -3.06 1.46E-08 1457356_at ------3.06 7.33E-05 1442820_at ------3.05 1.21E-06 1428304_at establishment of cohesion 1 homolog Esco2 -3.04 4.45E-06 1443777_at ------3.04 5.91E-07 1420081_s_at DNA segment, Chr 2, ERATO Doi 75D2Ertd750 -3.04 7.16E-07 1416961_at budding uninhibited by benzimidazol Bub1b -3.03 1.32E-05 1420082_at DNA segment, Chr 2, ERATO Doi 75D2Ertd750 -3.02 1.08E-06 1417822_at DNA segment, Chr 17, human D6S56D17H6S56 -3.02 9.49E-05 1442572_at ------3.02 1.80E-04 1452220_at dedicator of cytokinesis 1 Dock1 -3.02 1.98E-05 1421511_at Itgb3 -3.01 5.49E-07 1444615_x_at runt-related transcription factor 1; tra Runx1t1 -3.00 3.93E-07 1423520_at Lmnb1 -2.98 2.15E-09 1421963_a_at cell division cycle 25 homolog B (S. Cdc25b -2.98 1.92E-06 1428535_at RIKEN cDNA 9430020K01 gene 9430020K0 -2.97 8.08E-04 1450156_a_at hyaluronan mediated motility receptoHmmr -2.97 1.44E-05 1419486_at Foxc1 -2.97 4.22E-08 1422430_at fidgetin-like 1 Fignl1 -2.94 1.56E-05 1424118_a_at SPC25, NDC80 kinetochore complexSpc25 -2.94 3.47E-05 1419944_at Cyclin B1 Ccnb1 -2.93 1.30E-06 1427541_x_at hyaluronan mediated motility receptoHmmr -2.93 2.27E-05 1432582_at RIKEN cDNA 3110054G05 gene 3110054G0 -2.93 9.21E-04 1422264_s_at Kruppel-like factor 9 Klf9 -2.91 1.24E-04 1428294_at zinc finger protein 259 Zfp259 -2.91 2.16E-07 1419387_s_at mucin 13, epithelial transmembrane Muc13 -2.91 1.89E-05 1448576_at interleukin 7 receptor Il7r -2.88 4.85E-05 1423920_at non-SMC condensin I complex, subu Ncaph -2.88 8.16E-05 1436808_x_at minichromosome maintenance deficieMcm5 -2.86 1.95E-05 1449802_x_at RNA doma Rpusd2 -2.86 5.05E-04 1449030_at synapsin II Syn2 -2.85 3.35E-08 1455904_at growth arrest specific 5 /// small nucl Gas5 /// Sn -2.84 5.73E-06 1426071_at TGF-beta1-induced anti-apoptotic facTiaf2 -2.84 3.06E-04 1453596_at inhibitor of DNA binding 2 Id2 -2.84 9.62E-07 1457851_at ------2.83 4.99E-05 1441946_at inter-alpha (globulin) inhibitor H5 Itih5 -2.83 1.65E-05 1443552_at RIKEN cDNA E230008N13 gene E230008N -2.83 6.34E-06 1436763_a_at Kruppel-like factor 9 Klf9 -2.83 7.23E-06 1429172_a_at non-SMC condensin I complex, subu Ncapg -2.82 7.33E-07 1419695_at ST8 alpha-N-acetyl-neuraminide alphSt8sia1 -2.81 2.47E-05 1436203_a_at RIKEN cDNA 1110059G02 gene 1110059G0 -2.81 9.02E-07 1436204_at RIKEN cDNA 1110059G02 gene 1110059G0 -2.80 1.52E-05 1442833_at DNA segment, Chr 15, ERATO Doi 3D15Ertd30 -2.80 2.40E-06 1436368_at solute carrier family 16 (monocarbox Slc16a10 -2.79 1.18E-06 1427365_at 86 Krt86 -2.78 2.91E-08 1417574_at chemokine (C-X-C motif) ligand 12 Cxcl12 -2.77 3.88E-04 1441799_at RIKEN cDNA 6030422H21 gene 6030422H2 -2.76 1.13E-07 1435987_x_at RIKEN cDNA 1110059G02 gene 1110059G0 -2.75 7.34E-06 1440290_at predicted gene 10010 Gm10010 -2.75 4.20E-06 1440363_at ------2.74 3.67E-04 1438288_x_at RIKEN cDNA 1110059G02 gene 1110059G0 -2.73 1.49E-06 1451827_a_at NADPH oxidase 4 Nox4 -2.72 1.01E-05 1421088_at glypican 4 Gpc4 -2.72 5.74E-04 1453107_s_at RIKEN cDNA 4933413G19 gene /// f4933413G1 -2.72 1.05E-04 1422814_at asp (abnormal spindle)-like, microcepAspm -2.72 3.50E-06 1418776_at guanylate-binding protein 8 Gbp8 -2.71 1.57E-06 1442000_at major facilitator superfamily domain Mfsd2b -2.70 2.49E-04 1434592_at solute carrier family 16 (monocarbox Slc16a10 -2.70 1.14E-05 1460230_at synapsin II Syn2 -2.69 9.95E-06 1438527_at predicted gene 12816 /// predicted ge Gm12816 / -2.69 9.33E-06 1434767_at expressed sequence C79407 C79407 -2.69 2.61E-07 1451246_s_at aurora kinase B Aurkb -2.69 2.46E-08 1459429_at kinectin 1 Ktn1 -2.69 9.97E-04 1422498_at antigen, family H, 1 Mageh1 -2.68 4.51E-06 1437370_at shugoshin-like 2 (S. pombe) Sgol2 -2.67 5.65E-07 1418536_at histocompatibility 2, Q region locus 7H2-Q7 -2.67 4.74E-06 1429656_at Rho-related BTB domain containing Rhobtb1 -2.66 1.19E-04 1435393_at 1 receptor Mc1r -2.65 1.42E-04 1423626_at Dst -2.65 6.09E-07 1424128_x_at aurora kinase B Aurkb -2.65 3.43E-04 1415874_at similar to sprouty 1 /// sprouty homol LOC10004 -2.64 1.46E-04 1423635_at bone morphogenetic protein 2 Bmp2 -2.64 9.58E-06 1428481_s_at cell division cycle associated 8 Cdca8 -2.63 1.08E-06 1434748_at cytoskeleton associated protein 2 Ckap2 -2.63 8.93E-08 1423774_a_at protein regulator of cytokinesis 1 Prc1 -2.62 3.11E-07 1418021_at complement component 4B (Childo bC4b -2.61 2.56E-06 1438833_at cancer susceptibility candidate 5 Casc5 -2.61 1.96E-04 1416835_s_at S-adenosylmethionine decarboxylase Amd1 -2.60 8.81E-06 1453082_at tubby-like protein 3 Tulp3 -2.60 5.41E-08 1451691_at receptor type A Ednra -2.60 7.82E-05 1456328_at B-cell scaffold protein with r Bank1 -2.59 1.48E-07 1436755_at inter-alpha (globulin) inhibitor H5 Itih5 -2.58 4.24E-04 1458968_at expressed sequence C80283 C80283 -2.57 4.77E-04 1423753_at BMP and activin membrane-bound inBambi -2.57 4.68E-06 1429682_at family with sequence similarity 46, mFam46c -2.57 1.48E-07 1416442_at immediate early response 2 Ier2 -2.57 2.69E-04 1417635_at sperm autoantigenic protein 17 Spa17 -2.57 1.30E-04 1433525_at type A Ednra -2.56 4.15E-06 1459978_x_at cyclin J-like Ccnjl -2.56 1.94E-06 1423775_s_at protein regulator of cytokinesis 1 Prc1 -2.56 2.65E-07 1444089_at beta 2 Spnb2 -2.55 8.75E-07 1436966_at pellino 2 Peli2 -2.54 8.62E-06 1436847_s_at cell division cycle associated 8 Cdca8 -2.54 1.66E-06 1419406_a_at B-cell CLL/ 11A (zinc fingBcl11a -2.53 1.04E-05 1449171_at Ttk protein kinase Ttk -2.52 1.44E-05 1419153_at RIKEN cDNA 2810417H13 gene 2810417H1 -2.51 3.38E-05 1441189_at ------2.51 1.75E-04 1417784_at amyotrophic lateral sclerosis 2 (juvenAls2 -2.51 2.24E-05 1426818_at domain containing 4 Arrdc4 -2.50 1.13E-04 1459993_at ------2.50 1.15E-05 1419943_s_at cyclin B1 Ccnb1 -2.50 1.98E-06 1423100_at FBJ osteosarcoma oncogene Fos -2.48 3.16E-05 1448530_at guanosine monophosphate reductase Gmpr /// LO -2.47 7.52E-05 1457996_at ------2.46 2.34E-07 1446509_at spermine oxidase Smox -2.46 8.20E-08 1439178_at adrenergic receptor kinase, beta 2 Adrbk2 -2.45 1.51E-04 1429268_at RIKEN cDNA 2610318N02 gene 2610318N0 -2.45 3.74E-05 1452473_at proline rich 15 Prr15 -2.44 1.27E-06 1420271_at ------2.44 1.64E-04 1456341_a_at Kruppel-like factor 9 Klf9 -2.44 7.97E-06 1428105_at TPX2, microtubule-associated proteinTpx2 -2.43 2.11E-06 1423714_at ASF1 anti-silencing function 1 homo Asf1b -2.42 3.16E-06 1437176_at NLR family, CARD domain containinNlrc5 -2.42 9.63E-06 1417332_at regulatory factor X, 2 (influences HLRfx2 -2.41 8.86E-08 1421946_at C-reactive protein, pentraxin-related Crp -2.40 4.80E-06 1429159_at inter-alpha (globulin) inhibitor H5 Itih5 -2.40 8.07E-06 1455730_at discs, large (Drosophila) homolog-as Dlgap5 -2.40 3.20E-07 1415913_at predicted gene 15483 /// ribosomal prGm15483 / -2.40 8.33E-05 1439281_at solute carrier family 26, member 8 Slc26a8 -2.40 1.31E-04 1435336_at , EGF LAG seven-pass G-typCelsr2 -2.39 9.76E-04 1457072_at B-cell CLL/lymphoma 11A (zinc fingBcl11a -2.39 3.30E-05 1448205_at cyclin B1 Ccnb1 -2.39 7.78E-07 1437658_a_at small nucleolar RNA host gene (non-Snhg1 -2.38 1.29E-06 1444467_at expressed sequence C77534 C77534 -2.38 5.82E-04 1418982_at CCAAT/enhancer binding protein (C Cebpa -2.37 9.20E-05 1427141_at NDC80 homolog, kinetochore compl 2700099C1 -2.37 3.17E-06 1439819_at expressed sequence AU015263 AU015263 -2.36 2.83E-05 1456901_at a disintegrin-like and metallopeptidasAdamts20 -2.36 3.40E-04 1455983_at cell division cycle associated 2 Cdca2 -2.36 1.54E-04 1422016_a_at centromere protein H Cenph -2.35 1.46E-07 1419137_at SH3/ankyrin domain gene 3 Shank3 -2.35 3.35E-07 1434661_at synaptogyrin 1 Syngr1 -2.35 7.10E-05 1420691_at interleukin 2 receptor, alpha chain Il2ra -2.34 9.53E-08 1448688_at podocalyxin-like Podxl -2.34 2.24E-04 1421113_at pepsinogen 5, group I Pga5 -2.34 3.10E-06 1447494_at DNA segment, Chr 7, Brigham & WoD7Bwg082 -2.34 7.87E-05 1448325_at , regulatory (inhPpp1r15a -2.34 3.45E-06 1454744_at RIKEN cDNA F630043A04 gene F630043A0 -2.33 4.19E-05 1452459_at asp (abnormal spindle)-like, microcepAspm -2.33 2.56E-04 1446119_at ------2.33 8.73E-07 1415945_at minichromosome maintenance deficieMcm5 -2.32 5.22E-06 1420889_at holocytochrome c synthetase Hccs -2.32 3.69E-05 1416155_at high mobility group box 3 Hmgb3 -2.32 1.58E-06 1440762_at synapsin II Syn2 -2.32 1.21E-05 1437782_at contactin associated protein-like 2 Cntnap2 -2.31 4.10E-05 1419289_a_at synaptogyrin 1 Syngr1 -2.31 4.39E-04 1441964_at quaking Qk -2.30 7.86E-08 1429097_at ring finger protein 150 Rnf150 -2.29 9.07E-05 1439470_at ------2.29 1.03E-07 1423809_at transcription factor 19 Tcf19 -2.28 1.19E-05 1436211_at THO complex 4 Thoc4 -2.28 2.51E-05 1448650_a_at polymerase (DNA directed), epsilon Pole -2.27 4.81E-04 1442414_at Ring finger protein 103 Rnf103 -2.27 8.44E-07 1430983_at protein disulfide associatedPdia6 -2.27 4.56E-05 1459760_at NADH dehydrogenase (ubiquinone) FNdufs4 -2.26 1.72E-05 1424143_a_at chromatin licensing and DNA replicaCdt1 -2.26 5.16E-05 1428460_at synapsin II Syn2 -2.26 5.11E-04 1416129_at ERBB receptor feedback inhibitor 1 Errfi1 -2.26 7.92E-06 1431119_at DnaJ (Hsp40) related, subfamily B, mDnajb13 -2.26 5.16E-04 1417995_at protein tyrosine phosphatase, non-recPtpn22 -2.26 4.93E-09 1441083_at cleft lip and palate associated transmeClptm1 -2.25 2.19E-05 1441986_at zinc finger, CCHC domain containingZcchc6 -2.25 1.10E-04 1460292_a_at SWI/SNF related, matrix associated, Smarca1 -2.25 7.74E-04 1442263_at regulator of G-protein signaling 13 Rgs13 -2.24 2.40E-06 1442643_at KDM1 lysine (K)-specific demethylaKdm6b -2.24 4.77E-04 1433448_at solute carrier family 25, member 44 Slc25a44 -2.24 2.83E-07 1460515_at RIKEN cDNA 8430419K02 gene 8430419K0 -2.22 2.14E-06 1442941_at expressed sequence C77027 C77027 -2.22 6.29E-04 1454703_x_at small nucleolar RNA host gene (non-Snhg1 -2.22 3.32E-06 1437303_at signal transducer Il6st -2.22 3.09E-05 1424312_at receptor 1 Adipor1 -2.22 3.91E-07 1430476_at signal recognition particle 54B Srp54b -2.22 4.72E-06 1454182_at RIKEN cDNA 5430417C01 gene 5430417C0 -2.21 3.70E-04 1439912_at RIKEN cDNA 9430098F02 gene /// p9430098F0 -2.21 7.29E-05 1460241_a_at ST3 beta-galactoside alpha-2,3-sialyl St3gal5 -2.20 1.57E-04 1441698_at ------2.20 4.33E-04 1418285_at B1 Efnb1 -2.20 1.20E-04 1436882_at ubiquitin-like 5 Ubl5 -2.20 2.16E-07 1435710_at expressed sequence AI661384 AI661384 -2.19 4.35E-05 1452907_at Galc -2.19 1.13E-03 1459885_s_at cytochrome c oxidase, subunit VIIc //Cox7c /// L -2.18 5.73E-10 1425281_a_at TSC22 domain family, member 3 Tsc22d3 -2.18 1.22E-04 1446786_at ------2.17 1.01E-05 1437100_x_at proviral integration site 3 Pim3 -2.17 3.97E-05 1454832_at phosphatase and actin regulator 1 Phactr1 -2.16 1.37E-06 1438911_at septin 7 40428 -2.16 3.74E-06 1454903_at (TNFR sNgfr -2.16 7.07E-06 1428289_at Kruppel-like factor 9 Klf9 -2.16 3.74E-07 1420178_at ------2.16 1.32E-06 1434350_at cysteine-serine-rich nuclear protein 1 Csrnp1 -2.15 3.42E-04 1433891_at -rich repeat-containing G prot Lgr4 -2.13 1.04E-05 1426767_at WD repeat domain 90 Wdr90 -2.13 5.99E-04 1442347_at low density lipoprotein receptor-relat Lrp8 -2.13 1.61E-06 1439917_at PDZ domain containing 8 Pdzd8 -2.12 5.50E-04 1422398_at histone cluster 1, H1e Hist1h1e -2.12 1.50E-05 1449207_a_at kinesin family member 20A Kif20a -2.12 1.78E-05 1449147_at carbohydrate ( Gal-6) sChst1 -2.11 5.30E-04 1448037_at Zinc finger, CCHC domain containin Zcchc6 -2.11 3.10E-04 1452843_at interleukin 6 signal transducer Il6st -2.11 6.96E-04 1435293_at a disintegrin and metallopeptidase doAdam22 -2.11 1.13E-05 1434695_at denticleless homolog (Drosophila) Dtl -2.10 3.31E-06 1447643_x_at snail homolog 2 (Drosophila) Snai2 -2.10 9.51E-06 1437251_at cell division cycle associated 2 Cdca2 -2.10 2.03E-04 1439552_at triple functional domain (PTPRF inteTrio -2.09 6.49E-06 1427650_a_at runt related transcription factor 1 Runx1 -2.09 3.72E-07 1429481_at non-catalytic region of tyrosine kinas Nck2 -2.08 2.32E-07 1431301_at proline rich 15 Prr15 -2.08 3.45E-04 1452283_at Ras association (RalGDS/AF-6) dom Rassf8 -2.07 3.06E-04 1448484_at S-adenosylmethionine decarboxylase Amd1 -2.07 1.70E-06 1447434_at ------2.07 1.60E-05 1458502_at ------2.06 8.63E-05 1435396_at syntaxin binding protein 6 (amisyn) Stxbp6 -2.06 5.34E-06 1437716_x_at kinesin family member 22 Kif22 -2.06 5.63E-06 1444402_at zinc finger CCCH type containing 12Zc3h12c -2.06 7.94E-06 1422966_a_at Tfrc -2.06 3.93E-04 1426869_at biregional molecule-rel Boc -2.05 1.61E-06 1430955_at RIKEN cDNA 2810403A07 gene 2810403A0 -2.05 4.92E-04 1438076_at ------2.05 3.81E-07 1452592_at microsomal glutathione S- Mgst2 -2.05 5.84E-07 1428288_at Kruppel-like factor 9 Klf9 -2.05 6.54E-07 1448232_x_at predicted gene 3756 /// predicted gen Gm3756 /// -2.04 8.17E-05 1446562_at ------2.03 9.82E-08 1436909_at solute carrier family 25, member 44 Slc25a44 -2.03 2.45E-06 1444717_at ------2.03 3.85E-04 1429156_at RIKEN cDNA 2610036L11 gene 2610036L1 -2.02 1.38E-07 1446610_at engulfment and cell motility 1, ced-12Elmo1 -2.01 3.44E-06 1429165_at RIKEN cDNA 3110001I22 gene 3110001I22 -2.01 2.53E-04 1445298_at ------2.01 1.14E-03 1442629_at ------2.00 4.08E-05 1436202_at metastasis associated lung adenocarciMalat1 -2.00 8.71E-06 UP 1426175_a_at tryptase alpha/beta 1 Tpsab1 36.01 5.89E-08 1419717_at sema domain, immunoglobulin doma Sema3e 33.16 1.83E-09 1425967_a_at mast cell protease 4 Mcpt4 28.55 8.33E-10 1454959_s_at guanine nucleotide binding protein (GGnai1 20.17 1.61E-12 1448944_at 1 Nrp1 14.43 2.41E-07 1427038_at preproenkephalin Penk 14.34 1.07E-04 1417818_at WW domain containing transcription Wwtr1 13.09 2.27E-08 1438782_at contactin 4 Cntn4 12.79 3.95E-10 1448943_at neuropilin 1 Nrp1 12.40 1.18E-08 1427673_a_at sema domain, immunoglobulin doma Sema3e 10.73 8.75E-10 1429559_at guanine nucleotide binding protein, a Gnaq 8.38 1.32E-10 1460469_at receptor superfaTnfrsf9 8.05 3.75E-07 1428938_at guanine nucleotide binding protein, a Gnaq 7.89 8.60E-12 1424704_at runt related transcription factor 2 Runx2 7.77 7.13E-09 1420065_at Transketolase-like 1 Tktl1 7.42 1.42E-09 1458159_at ------7.41 7.31E-08 1455729_at guanine nucleotide binding protein, a Gnaq 7.32 2.25E-09 1417960_at cytoplasmic polyadenylation element Cpeb1 7.22 5.69E-09 1426873_s_at junction Jup 7.19 1.26E-11 1425814_a_at -like Calcrl 6.59 1.18E-10 1428940_at guanine nucleotide binding protein, a Gnaq 6.47 8.12E-07 1448664_a_at SPEG complex locus Speg 6.35 1.45E-08 1421688_a_at chemokine (C-C motif) ligand 1 Ccl1 6.14 5.92E-07 1428939_s_at guanine nucleotide binding protein, a Gnaq 6.09 5.28E-07 1450913_at UDP-Gal:betaGlcNAc beta 1,4-galac B4galt6 6.04 1.83E-06 1428034_a_at tumor necrosis factor receptor superfaTnfrsf9 5.81 7.10E-06 1437197_at sorbin and SH3 domain containing 2 Sorbs2 5.78 7.22E-08 1435622_at (glucosamine) 3-O-suHs3st3a1 5.77 7.52E-08 1455450_at protein tyrosine phosphatase, non-recPtpn3 5.69 9.35E-09 1420064_s_at transketolase-like 1 Tktl1 5.65 1.64E-07 1435758_at UDP-Gal:betaGlcNAc beta 1,4-galac B4galt6 5.63 3.30E-09 1460329_at UDP-Gal:betaGlcNAc beta 1,4-galac B4galt6 5.49 2.05E-07 1448213_at annexin A1 Anxa1 5.19 3.50E-08 1449567_at transketolase-like 1 Tktl1 5.11 7.59E-09 1435832_at leucine rich repeat containing 4 Lrrc4 5.05 1.67E-06 1449049_at toll-like receptor 1 Tlr1 4.85 9.18E-11 1425906_a_at sema domain, immunoglobulin doma Sema3e 4.47 1.49E-05 1435286_at Eph receptor A5 Epha5 4.47 2.29E-09 1418829_a_at 2, gamma neuronal Eno2 4.43 6.63E-08 1428914_at SH3 and PX domains 2A Sh3pxd2a 4.38 8.87E-08 1443518_at sarcoglycan, epsilon Sgce 4.28 6.27E-08 1459205_at ------4.25 1.18E-07 1449151_at cyclin-dependent kinase 18 Cdk18 4.18 8.66E-06 1450272_at tumor necrosis factor (ligand) superfaTnfsf8 4.00 3.93E-08 1420688_a_at sarcoglycan, epsilon Sgce 3.93 1.67E-10 1416097_at leucine rich repeat containing 4 Lrrc4 3.84 1.77E-11 1421304_at killer cell lectin-like receptor, subfamKlra2 3.79 3.83E-07 1452913_at Purkinje cell protein 4-like 1 Pcp4l1 3.77 2.32E-04 1419564_at zinc finger protein 467 Zfp467 3.75 2.89E-10 1436044_at , voltage-gated, type VScn7a 3.72 3.20E-10 1441727_s_at zinc finger protein 467 Zfp467 3.71 4.53E-10 1450237_at II beta Dnase2b 3.71 5.15E-09 1455849_at neuron navigator 1 Nav1 3.71 2.93E-07 1444690_at Eph receptor A5 Epha5 3.66 1.44E-06 1453721_a_at solute carrier family 31, member 2 Slc31a2 3.61 8.09E-09 1421256_at granzyme C Gzmc 3.59 9.78E-05 1422166_at C-type lectin domain family 2, membClec2i 3.59 3.58E-10 1436043_at sodium channel, voltage-gated, type VScn7a 3.57 6.38E-06 1446497_at ------3.55 3.74E-05 1416673_at beta-site APP-cleaving enzyme 2 Bace2 3.55 3.46E-09 1435407_at Zinc finger with KRAB and SCAN d Zkscan16 3.48 2.57E-07 1422444_at Itga6 3.48 1.22E-06 1418713_at pterin 4 alpha carbinolamine dehydraPcbd1 3.46 1.34E-09 1427442_a_at amyloid beta (A4) precursor protein App 3.45 9.36E-04 1441030_at retinoic acid induced 14 Rai14 3.45 9.84E-06 1454674_at fasciculation and elongation protein zFez1 3.42 2.71E-06 1442322_at ------3.41 3.06E-06 1435394_s_at ras homolog gene family, member C Rhoc 3.34 4.77E-08 1424769_s_at caldesmon 1 Cald1 3.33 2.64E-06 1457923_at ------3.31 1.69E-08 1432878_at RIKEN cDNA 4930544N03 gene 4930544N0 3.29 3.13E-05 1426260_a_at UDP glucuronosyltransferase 1 famil Ugt1a1 /// U 3.29 3.74E-04 1456426_at ------3.27 1.09E-07 1430386_at peptidylprolyl isomerase A pseudoge E030024N2 3.25 7.77E-06 1438081_at mutated in colorectal cancers Mcc 3.23 8.70E-05 1440527_at ------3.18 4.27E-07 1437920_at Eph receptor A5 Epha5 3.15 3.68E-09 1419329_at sorbin and SH3 domain containing 3 Sorbs3 3.14 1.44E-07 1437846_x_at beta-site APP-cleaving enzyme 2 Bace2 3.14 1.42E-07 1415797_at discoidin domain receptor family, meDdr1 3.11 5.98E-06 1458178_at ------3.10 4.74E-06 1416635_at , ac Smpdl3a 3.07 1.95E-07 1428861_at A interacting protein 1-like Filip1l 3.07 9.86E-08 1438466_at dynein, axonemal, heavy chain 7B Dnahc7b 3.07 1.24E-07 1420557_at Eph receptor A5 Epha5 3.07 2.05E-06 1425760_a_at phosphatidylinositol transfer protein, Pitpnm1 3.06 3.90E-07 1417932_at interleukin 18 Il18 3.05 2.75E-05 1448845_at P 25 subunit (human) Rpp25 3.03 2.81E-05 1418406_at phosphodiesterase 8A Pde8a 3.01 2.37E-09 1447616_at ------3.00 1.30E-07 1435857_s_at amyloid beta (A4) precursor-like protAplp1 2.97 2.61E-04 1442586_at suppressor of cytokine signaling 2 Socs2 2.93 1.46E-05 1433617_s_at UDP-Gal:betaGlcNAc beta 1,4-galac B4galt5 2.92 1.55E-06 1420859_at protein kinase inhibitor, alpha Pkia 2.91 1.76E-05 1424567_at 2 Tspan2 2.90 3.42E-06 1451475_at plexin D1 Plxnd1 2.89 1.17E-07 1420802_at interleukin 13 Il13 2.87 8.49E-07 1422138_at plasminogen activator, urokinase Plau 2.86 2.51E-04 1432606_at RIKEN cDNA 2610012C04 gene 2610012C0 2.85 9.32E-08 1424594_at sterile alpha motif domain containingSamd4 2.83 3.75E-07 1416654_at solute carrier family 31, member 2 Slc31a2 2.82 1.17E-08 1457491_at Pleckstrin homology domain containiPlekha1 2.81 7.32E-08 1455337_at FYVE, RhoGEF and PH domain contFgd4 2.80 1.23E-06 1436907_at neuron navigator 1 Nav1 2.78 6.25E-07 1446392_at ------2.77 2.49E-04 1458140_at slit homolog 2 (Drosophila) Slit2 2.77 6.38E-05 1429418_at CDC14 cell division cycle 14 homoloCdc14b 2.75 1.30E-06 1437190_at serine/threonine/tyrosine kinase 1 Styk1 2.75 1.23E-07 1417297_at inositol 1,4,5-triphosphate receptor 3 Itpr3 2.75 3.45E-06 1424659_at slit homolog 2 (Drosophila) Slit2 2.74 1.65E-05 1440650_at ------2.73 9.29E-07 1417128_at pleckstrin homology domain containi Plekho1 2.69 4.65E-06 1428058_at AHNAK (desmoyokin)Ahnak 2.67 2.66E-05 1419978_s_at DNA segment, Chr 10, ERATO Doi 6D10Ertd61 2.67 4.48E-05 1448547_at Ras association (RalGDS/AF-6) dom Rassf3 2.66 8.99E-08 1444542_at ------2.66 1.49E-05 1415892_at phosphate 1 Sgpl1 2.66 7.82E-10 1457214_at ------2.66 2.88E-06 1448892_at dedicator of cytokinesis 7 Dock7 2.62 5.41E-06 1422711_a_at pregnancy upregulated non-ubiquitouPnck 2.59 1.66E-06 1421600_a_at tripartite motif-containing 26 Trim26 2.58 1.75E-05 1441482_at ------2.57 3.76E-04 1433883_at 4 Tpm4 2.57 3.14E-09 1448596_at solute carrier family 6 (neurotransmitSlc6a8 2.55 1.07E-07 1460251_at Fas (TNF receptor superfamily membFas 2.55 4.91E-09 1426641_at tribbles homolog 2 (Drosophila) Trib2 2.54 1.61E-05 1429207_at RIKEN cDNA 5730408K05 gene 5730408K0 2.54 4.57E-09 1434399_at UDP-N-acetyl-alpha-D-galactosamin Galnt6 /// L 2.53 5.29E-06 1440721_at RIKEN cDNA 5930433N17 gene 5930433N1 2.52 2.93E-05 1434314_s_at RAB11 family interacting protein 5 ( Rab11fip5 2.52 3.74E-07 1428667_at monoamine oxidase A Maoa 2.52 3.99E-06 1458039_at ------2.51 3.20E-09 1459846_x_at canopy 2 homolog () Cnpy2 2.50 2.09E-06 1429281_at RIKEN cDNA 2610008E11 gene 2610008E1 2.50 6.12E-07 1449164_at CD68 antigen Cd68 2.49 1.89E-06 1451832_at chemokine-like factor Cklf 2.49 9.67E-05 1455143_at neuroligin 2 Nlgn2 2.48 1.68E-06 1438027_at ------2.48 7.53E-05 1446675_at adenosine kinase Adk 2.46 3.29E-06 1451525_at Rho GTPase activating protein 12 Arhgap12 2.46 8.43E-04 1447886_at RIKEN cDNA 0610040B09 gene 0610040B0 2.45 2.24E-04 1424542_at S100 calcium binding protein A4 S100a4 2.45 7.99E-05 1417471_s_at DNA segment, Chr 1, ERATO Doi 62D1Ertd622 2.44 1.50E-07 1441576_at ------2.43 3.04E-05 1418407_at phosphodiesterase 8A Pde8a 2.42 1.08E-03 1456593_at ------2.42 1.11E-04 1418809_at paired-Ig-like receptor A2 Pira2 2.39 8.10E-07 1421605_a_at aquaporin 9 Aqp9 2.38 9.50E-05 1451474_a_at poly (ADP-ribose) polymerase familyParp8 2.37 4.62E-07 1459744_at CD53 antigen Cd53 2.36 1.56E-07 1438366_x_at 3 Clcn3 2.35 2.61E-05 1426640_s_at tribbles homolog 2 (Drosophila) Trib2 2.34 1.62E-05 1447167_at ------2.34 1.02E-05 1418483_a_at galactosyltransferase alpGgta1 2.33 1.60E-06 1435271_at regulatory factor 3 Irf3 2.32 1.93E-06 1455175_at PHD finger protein 13 Phf13 2.32 7.43E-09 1459595_at ------2.32 5.69E-09 1435402_at GRAM domain containing 1B Gramd1b 2.31 1.09E-07 1431980_a_at arsenic (+3 oxidation state) methyltraAs3mt 2.31 3.30E-07 1425315_at dedicator of cytokinesis 7 Dock7 2.31 5.98E-07 1448721_at DNA segment, Chr 1, ERATO Doi 62D1Ertd622 2.30 9.27E-07 1421309_at O-6-methylguanine-DNA methyltransMgmt 2.30 2.05E-08 1454933_at family with sequence similarity 176, Fam176b 2.30 1.48E-08 1449187_at platelet derived growth factor, alpha Pdgfa 2.29 5.11E-07 1432417_a_at tetraspanin 2 Tspan2 2.29 1.00E-06 1459362_at ------2.28 4.76E-04 1417903_at deafness, autosomal dominant 5 (humDfna5 2.27 4.53E-09 1436636_at ------2.27 2.36E-07 1421594_a_at synaptotagmin-like 2 Sytl2 2.26 2.14E-07 1459238_at ------2.26 5.23E-07 1424133_at transmembrane protein 98 Tmem98 2.26 1.32E-06 1442157_at ------2.24 6.12E-05 1439937_at Transmembrane 9 superfamily membTm9sf1 2.24 7.69E-06 1447962_at ------2.24 1.33E-06 1458188_at ------2.24 1.27E-04 1427405_s_at RAB11 family interacting protein 5 ( Rab11fip5 2.23 4.27E-04 1445370_at ------2.23 1.15E-06 1449688_at DNA segment, Chr 10, ERATO Doi 6D10Ertd61 2.22 3.91E-04 1441589_at ------2.21 1.20E-04 1450436_s_at DnaJ (Hsp40) homolog, subfamily B,Dnajb5 2.21 1.91E-05 1450355_a_at capping protein (actin filament), gelsoCapg 2.20 1.71E-08 1438800_at N-acetylglucosamine kinase Nagk 2.20 2.00E-04 1443162_at ------2.19 1.78E-04 1437070_at CDC14 cell division cycle 14 homoloCdc14b 2.19 1.63E-07 1429831_at phosphoinositide-3-kinase adaptor pr Pik3ap1 2.19 6.32E-05 1447315_at ------2.18 1.05E-04 1444752_at RIKEN cDNA C230073G13 gene C230073G 2.17 9.02E-04 1455615_at chemokine-like factor Cklf 2.17 7.91E-05 1434291_a_at small EDRK-rich factor 1 Serf1 2.17 1.03E-05 1451486_at solute carrier family 46, member 3 Slc46a3 2.16 7.58E-06 1445105_at ------2.16 1.28E-04 1421598_at ------2.15 2.51E-06 1417558_at Fyn proto-oncogene Fyn 2.14 7.47E-06 1435692_at tetramerisation do Kctd21 2.13 1.03E-05 1442800_x_at family with sequence similarity 181, Fam181b 2.13 3.97E-04 1460457_at RIKEN cDNA 2810405F17 gene 2810405F1 2.13 6.60E-04 1435461_at membrane associated guanylate kinasMagi3 2.12 1.78E-04 1424970_at purine-rich element binding protein GPurg 2.12 5.14E-09 1444613_at ------2.11 1.14E-04 1437724_x_at phosphatidylinositol transfer protein, Pitpnm1 2.11 5.63E-08 1452487_x_at paired-Ig-like receptor A2 Pira2 2.11 3.03E-05 1425769_x_at chemokine-like factor Cklf 2.10 1.33E-05 1437477_at leucine rich repeat (in FLII) interactinLrrfip1 2.10 5.97E-09 1423602_at TNF receptor-associated factor 1 Traf1 2.10 7.96E-06 1424568_at tetraspanin 2 Tspan2 2.10 5.77E-08 1452847_at GATS protein-like 3 Gatsl3 2.09 2.67E-05 1441418_at ------2.09 1.14E-03 1417327_at caveolin 2 Cav2 2.08 5.96E-05 1459649_at ------2.07 9.31E-04 1440304_at ------2.06 4.19E-06 1433558_at disabled homolog 2 (Drosophila) inteDab2ip 2.06 1.13E-06 1458501_at vesicle-associated membrane protein,Vapb 2.06 5.31E-05 1416473_a_at immunoglobulin superfamily, DCC suIgdcc4 2.05 2.90E-05 1441370_at Transmembrane and coiled coil domaTmcc1 2.05 6.15E-05 1427424_at UDP-N-acetyl-alpha-D-galactosamin Galnt6 /// L 2.05 4.83E-06 1448765_at Fyn proto-oncogene Fyn 2.05 1.23E-06 1446911_at ------2.05 7.57E-05 1420464_s_at predicted pseudogene 10693 /// predi Gm10693 / 2.04 3.45E-06 1449602_at ------2.04 4.95E-06 1442073_at inositol polyphosphate-1-phosphataseInpp1 2.04 1.36E-04 1453016_at heat shock B (small), mHspb11 2.04 5.38E-07 1418072_at histone cluster 1, H2bc Hist1h2bc 2.03 3.85E-08 1421444_at receptor Pgr 2.03 7.49E-05 1460696_at cDNA sequence BC026585 BC026585 2.03 1.31E-06 1444777_at Retinoic acid induced 14 Rai14 2.01 7.28E-05 1445460_at ------2.01 9.98E-04 1442605_at ------2.01 1.43E-04 1429671_at SCAN domain containing 3 Scand3 2.00 1.68E-04 A 1500 )

6 IL-3 IL-3+SCF ) 6 40 1000 30

20

10

Cell number (cell X10 (cell number Cell 0 500 0 2 4 6 8 Weeks Cell number (cell X10 0 0 1 2 3 4 5 6 7 8 9 Weeks

B IL-3-cultured cells C IL-3+SCF-cultured cells

128 128

96 96

64 64 Events Events Events 32 32

0 1 2 3 4 10 10 10 10 0 101 102 103 104 FcεRI FcεRI

128 128

96 96

64 64 Events Events Events 32 32

0 1 2 3 4 10 10 10 10 0 101 102 103 104 KIT KIT D

Culture conditions Stain x40 x100

IL-3

Toluidine blue IL-3+SCF

E

IL-3

Safranin/ Alcian blue

IL-3+SCF

10 μm

Figure S1 Culture conditions Stimulus A x40 x100

Control

IL-3

Ag

B

Control

IL-3+SCF

Ag

10 μm

Figure S2 A x40 x100 Stain

Toluidine blue

B

Safranin/ Alcian blue

10 μm

C Released cytokines

3000

2000

IL-6 (pg/ml) 1000 * * 0 * * * Ag (10 ng/ml) - + + + + - - + + + + - SCF (ng/ml)

D Retained cytokines

300

200

IL-6 (pg/ml) 100

0 * * * * Ag (10 ng/ml) - + + + + - - + + + + - SCF (ng/ml)

Figure S3