Int. J. Mol. Sci. 2013, 14, 23516-23532; doi:10.3390/ijms141223516 OPEN ACCESS

International Journal of

Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article

Transcriptional Profiling of Hilar Nodes from Pigs after Experimental Infection with Actinobacillus Pleuropneumoniae Shumin Yu 1,2, Zhicai Zuo 1,2, Hengmin Cui 1,2,*, Mingzhou Li 3, Xi Peng 1,2, Ling Zhu 1,2, Ming Zhang 3, Xuewei Li 3, Zhiwen Xu 1,2, Meng Gan 1,2, Junliang Deng 1,2, Jing Fang 1,2, Jideng Ma 3, Shengqun Su 4, Ya Wang 1,2, Liuhong Shen 1,2, Xiaoping Ma 1,2, Zhihua Ren 1,2, Bangyuan Wu 1,2 and Yanchun Hu 1,2 1

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College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, China; E-Mails: [email protected] (S.Y.); [email protected] (Z.Z.); [email protected] (X.P.); [email protected] (L.Z.); [email protected] (Z.X.); [email protected] (M.G.); [email protected] (J.D.); [email protected] (J.F.); [email protected] (Y.W.); [email protected] (L.S.); [email protected] (X.M.); [email protected] (Z.R.); [email protected] (B.W.); [email protected] (Y.H.) Laboratory of Animal Disease and Human Health, Sichuan Agricultural University, Ya’an 625014, China College of Animal Science and Technology, Sichuan Agricultural University, Ya’an 625014, China; E-Mails: [email protected] (M.L.); [email protected] (M.Z.); [email protected] (X.L.); [email protected] (J.M.) Library of Sichuan Agricultural University, Ya’an 625014, China; E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-136-0826-4628; Fax: +86-835-2882340. Received: 24 September 2013; in revised form: 12 November 2013 / Accepted: 15 November 2013 / Published: 29 November 2013

Abstract: The gram-negative bacterium Actinobacillus pleuropneumoniae (APP) is an inhabitant of the porcine upper respiratory tract and the causative agent of porcine pleuropneumonia (PP). In recent years, knowledge about the proinflammatory cytokine and chemokine gene expression that occurs in lung and lymph node of the APP-infected swine has been advanced. However, systematic gene expression profiles on hilar nodes from pigs after infection with Actinobacillus pleuropneumoniae have not yet been reported. The transcriptional responses were studied in hilar nodes (HN) from swine experimentally infected with APP and the control groupusing Agilent Porcine Genechip, including 43,603 probe sets. 9,517 transcripts were identified as differentially expressed (DE) at the

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p ≤ 0.01 level by comparing the log2 (normalized signal) of the two groups named treatment group (TG) and controls (CG). Eight hundred and fifteen of these DE transcripts were annotated as pig genes in the GenBank database (DB). Two hundred and seventy-two biological process categories (BP), 75 cellular components and 171 molecular functions were substantially altered in the TG compared to CG. Many BP were involved in host immune responses (i.e., signaling, signal transmission, signal transduction, response to stimulus, oxidation reduction, response to stress, immune system process, signaling pathway, immune response, cell surface receptor linked signaling pathway). Seven DE gene pathways (VEGF signaling pathway, Long-term potentiation, Ribosome, Asthma, Allograft rejection, Type I diabetes mellitus and Cardiac muscle contraction) and statistically significant associations with host responses were affected. Many cytokines (including NRAS, PI3K, MAPK14, CaM, HSP27, protein phosphatase 3, catalytic subunit and alpha isoform), mediating the proliferation and migration of endothelial cells and promoting survival and vascular permeability, were activated in TG, whilst many immunomodulatory cytokines were suppressed. The significant changes in the expression patterns of the genes, GO terms, and pathways, led to a decrease of antigenic peptides with antigen presenting cells presented to T lymphocytes via the major histocompatibility complex, and alleviated immune response induced APP of HN. The immune response ability of HN in the APP-infected pigs was weakened; however, cell proliferation and migration ability was enhanced. Keywords: porcine pleuropneumonia; infection; Actinobacillus pleuropneumoniae; Agilent Porcine Genechip; microarray analyses; cytokine; host defense response

1. Introduction Members of the Actinobacillus genus (family Pasteurellaceae) are Gram-negative facultative anaerobic bacteria. They range from commensal species of the mammalian respiratory and genital tracts to important pathogens [1,2]. Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia (PP), a disease occurring worldwide and causing significant economic losses in the swine industry [3–7]. The disease, which occurs in swine of all ages, is highly infectious, often fatal, and characterized by necrotizing, hemorrhagic bronchopneumonia and serofibrinous pleuritis [8]. APP can spread quickly by air-borne particles and/or touching contaminated surface, and often kill infected animals in the acute phase when extensive lung hemorrhage and necrosis occur. Swine that survived often developed pleurisy, the sequelaes of local necrosis of the pleura, or became healthy carriers of APP. APP can be divided into two biovars based on its nicotinamide adenine dinucleotide (NAD) requirements: biovar 1, which is NAD dependent, and biovar 2, which can synthesize NAD in the presence of specific pyridine nucleotides or their precursors [4]. The porcine lung infected with APP has previously been reported to result in local production of proinflammatory proteins or to mRNA encoding the cytokines interleukin (IL)-1α, IL-1β, IL-6 and the chemokine IL-8 [9,10]. Likewise, bioactive protein and/or mRNA coding for IL-10, IL12p35, TNF-α and IFN-α had been shown to be up-regulated after infection with APP in vivo or in vitro [9–15].

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Using cDNA microarrays, Moser and co-workers identified 307 anonymous transcripts in blood leukocytes obtained from pigs that were severely affected by experimental infection with APP [16]. Hedegaard et al. investigated the molecular characterization of the early response in pigs to infection with APP serotype 5B, using cDNA microarrays [17]. In this study, Hedegaard et al. found three subsets of genes that were consistently expressed at different levels depending upon the infection status and a total of 130 genes had different expression profiles in hilar node (HN) tissue samples from infected versus non-infected animals [17]. Mortensen et al. studied the local transcriptional response in different locations of lung from pigs experimentally infected with the respiratory pathogen APP 5B, using porcine cDNA microarrays (DJF Pig 55 K v1) representing approximately 20,000 porcine genes printed in duplicate [18]. Within the lung, Mortensen et al. found a clear division of induced genes as, in unaffected areas a large part of differently expressed genes were involved in systemic reaction to infection, while differently expressed genes in necrotic areas were mainly concerned with homeostasis regulation [18]. Zuo et al. studied the relationship between infection and injury by investigating the whole porcine genome expression profiles of swine lung tissues post-inoculated with experimental APP, and found 11,929 transcripts differentially expressed at the p ≤ 0.01 level [19]. Many proinflammatory-inflammatory cytokines were activated and involved in the regulation of the host defense response at the site of inflammation; while the cytokines involved in regulation of the host immune response were suppressed [19]. APP often kill infected swine in the acute phase, the lung and pleural of dead animals character by necrotizing, hemorrhagic bronchopneumonia and serofibrinous pleuritis. HN is one of the important immune organs adjacent to lung. Thus, transcriptional profiling of whole porcine genome in HN sampled from inoculated versus non-inoculated swine would lead to greater knowledge of the host response dynamics to bacterial infection in the lung. This knowledge is important to obtain a more complete picture of the lung-specific host reaction in the pathogenesis of respiratory infection. In the present study, the Agilent Whole Porcine Genome Oligo Microarrays were used to detect the changes in gene expression of infected pigs’ HN from non-inoculated animals. Ten transcripts (the top six up-regulated and the top four down-regulated in microarray data) were selected to verify the accuracy and reproducibility of the microarray data by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). 2. Results 2.1. Clinical Symptoms and Necropsy Findings Swines showed hyperthermia (40.6–42.0 °C), dyspnea and anorexia after being inoculated with APP 24–48 h. Two swines died with respiratory distress at post-inoculation 36–48 h. In the autopsy, the lungs were severely damaged by acute, multifocal, fibrino-necrotizing and hemorrhagic pneumonia complicated by acute diffuse fibrinous pleuritis. The HN were enlarged and congested. No lesions in CG were observed. The infected swines had lung and pleural lesions of variable severity consistent with acute pleuropneumonia, whereas the surrounding lung and pleural tissue appeared normal.

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Histopathological lesions were not observed in CG. However, lesions in TG’s in TG’s HN were characterized by loose medulla, congestion, edema, fibrinous exudation and neutrophils infiltration (Figure 1). Figure 1. No lesions were observed in control group (CG) hilar nodes (HN) (A) (scale bar = 25 μm, 400×); The HN in treatment group (TG), the little veins are congested in medulla (B) (scale bar = 25 μm, 400×); and the little veins and capillaries are congested in cortex and medulla (C) (scale bar = 25 μm, 400×); and the medulla are obvious edema, and neutrophils infiltration (D) (scale bar = 25 μm, 400×).

2.2. Microarray Profiling Expression profiling was conducted using a commercially available Agilent Porcine Genechip that included 43,603 probe sets. The transcriptome of the HN was determined. Expression was detected for 29,105 probes (66.75% of all probe sets) of the CG. A total of 29,646 probes (67.99% of all probe sets) were expressed in TG. When probe sets’ intensities had been normalized and filtered, there were still 26,353 probes used to identify significantly DE genes. There were 9,517genes identified as DE at the p ≤ 0.01 level by comparing the log2 (normalized signal) of the two groups (CG vs. TG) using t-test analysis. Hierarchical clustering was applied to the mean log-ratio of the replicated spots from the DE genes by the average linkage and used euclidean distance as the similarity metric (Figure 2). The expression profiles of samples were divided into two groups- one from the non-inoculated swines (M-L-1, M-L-2, M-L-3) and the other group from the inoculated swine (M-L-4, M-L-5, M-L-6).

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Figure 2. Hierarchical clustering analysis and clustering segmentation.

The principal component map to three-dimensional space, also found that the distance of CG (samples M-L-1, M-L-2, M-L-3) is short, and the gene expression pattern is more consistent. The distance of TG (samples M-L-4, M-L-5, M-L-6) is relatively discrete because of the differences in the lesions degree. The gene expression pattern is similar (Figure 3). Figure 3. Three-dimensional map of principal component analysis (PCA) for mapping samples obtained from clustering segmentation.

The six samples were set as variables. The principal component analysis (PCA) of the co-expressed differentially genes (CG vs. TG) showed that the contribution rate of the first principal component of which has reached 97.16%. The total contribution rate of the first three principal components reached 99.41% (Table 1).

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Table 1. Eigenvalues and contribution ratio of PCA for differential expression genes. Principal Component 1 2 3 4 5 6

Eigenvalues 42.273 0.978 0.147 0.072 0.025 0.014

Contribution ratio 97.16% 2.25% 0.34% 0.17% 0.06% 0.03%

2.3. Differentially Expressed (DE) Gene Analysis Of the 9,517 DE genes, 815 were annotated as pig genes in the GenBank database (DB). GO and KEGG pathway analyses of the 815 DE gene lists were conducted using DAVID. There were 272 biological process (BP) categories (Supplementary Table S1), 75 cellular components (Supplementary Table S2), and 171 molecular functions (Supplementary Table S3) were significantly affected by infection with APP (p = 0). There were many BP related to the immune responses, and a number of BP related to metabolism were also identified. A total of 344 genes were analyzed by pathway enrichment analysis (GSEA). Two hundred and thirty-eight genes (69.19% of the total) were correlated with TG, while the other 106 (30.81%) genes were correlated with CG. One hundred and forty-two of the total 170 pathways remained for further analysis after size filtering (2 ≤ sizes ≤ 20). Altogether, 104/142 pathways (Supplementary Table S4) were enriched and up-regulated in the TG and down-regulated in the CG. Two pathways (namely, VEGF signaling pathway and Long-term potentiation) were significantly enriched at nominal p values of less than 1% and 5%. Thirty-eight pathways (Supplementary Table S5) were down-regulated in the TG. One pathway (Allograft rejection) was statistically significant at a false discovery rate of 6.0. Labeling and hybridization of the cRNA was performed with Agilent Whole Porcine Genome Oligo (4 × 44K) Microarrays (one-color platform) at the National Engineering Center for Biochip at Shanghai, according to the manufacturer’s protocols. The slides were scanned and analyzed using the histogram method with default settings in an Agilent G2565AA and Agilent G2565BA Microarray Scanner System using SureScan Technology. The TIFF image generated was loaded into Feature Extraction Software (Agilent Technologies, Sisha Clara, CA, USA) for feature data extraction, and data analysis was performed with MultiExperiment Viewer (MeV) (Boston, MA, USA). The array data has been submitted to gene expression omnibus (GEO) GSE42317 [50]. Comparisons between the CG and the TG were conducted using three biological replicates for each group. CG samples and TG samples were used for microarray analysis. The six Microarray data were normalized using the quantile method [51] with WebarrayDB online microarray data analysis [52]. Data were filtered and assessed by the MIDAW online analysis program [53] using the method of weighted K-nearest neighbor [54]. T-tests for microarray data were performed by a MultiExperiment Viewer (MeV) software package (Version 4.5, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA, USA) [55]. Tests for statistical significance (p < 0.05), overrepresentation of Gene ontology (GO) terms [56,57], and pathway in Kyoto Encyclopedia of Genes and Genomes (KEGG) DB [58,59] among both induced and repressed genes were conducted using the ErmineJ gene set analysis software [60] and the Database for Annotation, Visualization and Integrated Discovery (DAVID) Online platform with a threshold of a minimum three genes annotated at each node. The leading edge analysis for the pathway of differential expression in microarray data with a threshold of a minimum of two genes and maximum of 20 genes annotated at each node were conducted using the gene set enrichment analysis (GSEA) V2.06 package at the GSEA/MSigDB web site (the broad institute) [61–63]. More detailed descriptions of the microarray experiments are available at the NCBI Gene Expression Omnibus [64–66]. 4.3. Real-Time QRT-PCR In order to confirm reliability of the expression profile in the microarray analyses, expression level of the genes (six up-regulated and four down-regulated) were performed by real-time qRT-PCR. Sequences for the primers were obtained from Genbank and NCBI. Primers were designed using the software Primer 5 and synthesized at Invitrogen Co. Led (Shanghai, China) (Table 2). Extracted RNA was converted into cDNA by reverse transcription of 1 μL total RNA using SYBR® PrimeScript™ RT-PCR Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol, and then cDNA was stored at −20 °C until use. Quantitative PCR was performed in a 25 μL reaction volume (2 μL cDNA, 12.5 μL of SYBR® Premix Ex TaqTM (2×) TaKaRa, Shiga, Japan), 0.5 μL of 10 μM upstream and downstream primers respectively, and added ddH2O to 25 μL) on the BIO-RAD IQ5 System (BIO-RAD, Hercules, CA, USA). Real-time PCR conditions were as follows: 30 s at 95.0 °C, 40 cycles

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of denaturation at 95 °C for 5 s followed by 30 s annealing and elongation at 51.2–60 °C (Table 2). Information of primer pairs is reported in Table 2. Melting curves were obtained at the end of each run to confirm a single PCR product. All samples were run in triplicate. Non-template controls were included in each run to exclude contamination and nonspecific amplification. Expression levels of samples were normalized by using a normalization factor calculated by the program geNorm. This normalization factor was calculated based on RT-qPCR results for three selected reference genes, ACTB, TOP2B and TBP. Table 2. Information on the primers used for QRT-PCR. Confirmation objects

Gene symbol ACTB

Reference gene

TBP TOP2B RETN ADAM17 GPNMB

Up gene CHRM1 ALDH2

Primer sequence (5'→3') TCTGGCACCACACCTTCT TGATCTGGGTCATCTTCTCAC GATGGACGTTCGGTTTAGG AGCAGCACAGTACGAGCAA AACTGGATGATGCTAATGATGCT TGGAAAAACTCCGTATCTGTCTC AGTGCGCTGGCATAGACTGG CATCCTCTTCTCAAGGTTTATTTCC TTGAGGAAGGGGAAGCC ACGGAGCCCACGATGTT GAGACCCAGCCTTCCTT TTGCTTTCTATCGCTTTGTA CGCTGGTCAAGGAGAAGAA GCACATGGGGTTGATGGT AAACTGCTCTGCGGTGGA CGTACTTGGAATTGTTGGCTC GTCGAGGCTGTGCAGATTAG

IL6

Amplicon Ta (°C) length (bp)

GenBank No.

114

60

DQ178122

124

60

DQ178129

137

60

AF222921

197

60

NM_213783

158

56

NM_001099926

130

51.2

NM_001098584

185

56

NM_214034

181

56

NM_001044611

101

56

NM_214399

107

56

NM_213813

158

51.2

NM_213999

291

51.2

NM_001031796

176

56

NM_001105294

GCATTTGTGGTGGGGTTAG KLRK1 DUOX2 Down gene OAS2 KCNAB1

TGATGTGATAAACCGTGGTG TGGATCGGGCAAGGAAA CCCTTCTTCAACTCCCTG CAAAAGTTCTCATAGTGGTGC GACACGGCTGAAGGTTT TGGCACGTCCCAAGACT AAGGGAGAAAACAGCAAAAC AACCTGAATGGCACCGA

This allowed quantification of the target gene in one sample relative to that in another (the calibrator) using the “2−ΔΔCt method” of calculating fold changes in gene expression [67]. Correlation analysis between qRT-PCR and microarray was conducted.

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5. Conclusions We have generated reliable mRNA transcriptomes of HN from the APP-infected and negative control pigs and identified a set of DE genes in our current case-control study, and a functional enrichment analysis indicated that these DE genes were mainly related to “host immune response” and “host response”. We found that, in the APP-infected HN, many cytokineswere activated and involved in cell migration signaling induced response cells as granulocytes (e.g., neutrophils, eosinophils, and basophils) and monocytes migration to the inflammatory site, while the cytokines involved in regulation of the host immune response were suppressed. The current study provides data that can be used in future studies to decipher the molecular mechanism of the systematic influences from porcine pleuropneumonia. Acknowledgments We thank the farmer that provided swine. This work was supported financially by the Natural Science Foundation of Science and Technology Department of Sichuan Province. Conflicts of Interest The authors declare no conflict of interest. Our experiments involving the use of swine, and the use of pigs and all experimental procedures involving animals were approved by Sichuan Agricultural University Animal Care and Use Committee. References 1.

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Supplementary Information Table S1. The significant gene ontology biological processes in swines. Name [GO:0019538] [GO:0009058] [GO:0051179] [GO:0044249] [GO:0006810] [GO:0044267] [GO:0023052] [GO:0006807] [GO:0034641] [GO:0023060] [GO:0007165] [GO:0019222] [GO:0006139] [GO:0044281] [GO:0010467] [GO:0050896] [GO:0080090] [GO:0031323] [GO:0060255] [GO:0055114] [GO:0006464] [GO:0034645] [GO:0009889] [GO:0010468] [GO:0051171] [GO:0009056] [GO:0010556] [GO:0061019] [GO:0006508] [GO:0043687] [GO:0044248] [GO:0006355] [GO:0032502] [GO:0032501] [GO:0007275] [GO:0006796] [GO:0006629] [GO:0016310] [GO:0016043] [GO:0005975] [GO:0006950] [GO:0061018]

Describtion protein metabolic process biosynthetic process localization cellular biosynthetic process transport cellular protein metabolic process signaling nitrogen compound metabolic process cellular nitrogen compound metabolic process signal transmission signal transduction regulation of metabolic process cellular nucleobase, nucleoside, nucleotide and nucleic acid metabolic process small molecule metabolic process gene expression response to stimulus regulation of primary metabolic process regulation of cellular metabolic process regulation of macromolecule metabolic process oxidation reduction protein modification process cellular macromolecule biosynthetic process regulation of biosynthetic process regulation of gene expression regulation of nitrogen compound metabolic process catabolic process regulation of macromolecule biosynthetic process regulation of transcription proteolysis post-translational protein modification cellular catabolic process regulation of cellular transcription, DNA-dependent developmental process multicellular organismal process multicellular organismal development phosphate metabolic process lipid metabolic process phosphorylation cellular component organization carbohydrate metabolic process response to stress transcription

Probe Genes 92 92 80 80 73 73 73 73 70 70 67 67 63 63 58 58 55 55 53 53 51 51 50 50 48

48

48 48 47 46 46 46 45 44 43 42 42 41 41 41 40 38 36 34 33 33 33 31 31 30 28 27 27 27 27

48 48 47 46 46 46 45 44 43 42 42 41 41 41 40 38 36 34 33 33 33 31 31 30 28 27 27 27 27

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S2 Table S1. Cont.

Name [GO:0006811] [GO:0002376] [GO:0006082] [GO:0023033] [GO:0065008] [GO:0044283] [GO:0007242] [GO:0006468] [GO:0044255] [GO:0006955] [GO:0007166] [GO:0048856] [GO:0009057] [GO:0032787] [GO:0033036] [GO:0048869] [GO:0048731] [GO:0006091] [GO:0006812] [GO:0044265] [GO:0044262] [GO:0030154] [GO:0006996] [GO:0048518] [GO:0048519] [GO:0008219] [GO:0048523] [GO:0006631] [GO:0010941] [GO:0042592] [GO:0012501] [GO:0051186] [GO:0044271] [GO:0030163] [GO:0006066] [GO:0023034] [GO:0044257] [GO:0006952] [GO:0048513] [GO:0030001] [GO:0055086] [GO:0007186] [GO:0043632] [GO:0008610]

Describtion ion transport immune system process organic acid metabolic process signaling pathway regulation of biological quality small molecule biosynthetic process intracellular signaling cascade protein amino acid phosphorylation cellular lipid metabolic process immune response cell surface receptor linked signaling pathway anatomical structure development macromolecule catabolic process monocarboxylic acid metabolic process macromolecule localization cellular developmental process system development generation of precursor metabolites and energy cation transport cellular macromolecule catabolic process cellular carbohydrate metabolic process cell differentiation organelle organization positive regulation of biological process negative regulation of biological process cell death negative regulation of cellular process fatty acid metabolic process regulation of cell death homeostatic process programmed cell death cofactor metabolic process cellular nitrogen compound biosynthetic process protein catabolic process alcohol metabolic process intracellular signaling pathway cellular protein catabolic process defense response organ development metal ion transport cellular nucleobase, nucleoside and nucleotide metabolic process G-protein coupled receptor protein signaling pathway modification-dependent macromolecule catabolic process lipid biosynthetic process

Probe Genes 27 27 26 26 26 26 26 26 25 25 24 24 24 24 23 23 22 22 22 22 22 22 21 21 21 21 21 21 20 20 20 20 19 19 19 19 19 19 18 18 18 18 18 18 18 18 18 18 18 18 17 17 17 17 15 15 15 15 15 15 15 15 14 14 14 14 14 14 14 14 14 14 13 13 13 13 13 13 13 13 12 12 12 12 11 11 11 11

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S3 Table S1. Cont.

Name [GO:0008104] [GO:0046483] [GO:0015672] [GO:0007155] [GO:0048583] [GO:0044282] [GO:0051716] [GO:0042221] [GO:0051704] [GO:0007049] [GO:0045184] [GO:0048522] [GO:0015980] [GO:0009607] [GO:0045333] [GO:0006412] [GO:0007264] [GO:0019725] [GO:0046394] [GO:0009165] [GO:0000003] [GO:0009100] [GO:0009109] [GO:0048584] [GO:0032879] [GO:0002682] [GO:0010876] [GO:0019318] [GO:0009308] [GO:0022414] [GO:0008202] [GO:0009653] [GO:0019882] [GO:0033554] [GO:0009101] [GO:0016042] [GO:0044085] [GO:0002684] [GO:0006633] [GO:0016192] [GO:0009611] [GO:0051301] [GO:0031325] [GO:0006519]

Describtion protein localization heterocycle metabolic process monovalent inorganic cation transport cell adhesion regulation of response to stimulus small molecule catabolic process cellular response to stimulus response to chemical stimulus multi-organism process cell cycle establishment of protein localization positive regulation of cellular process energy derivation by oxidation of organic compounds response to biotic stimulus cellular respiration translation small GTPase mediated signal transduction cellular homeostasis carboxylic acid biosynthetic process nucleotide biosynthetic process reproduction glycoprotein metabolic process coenzyme catabolic process positive regulation of response to stimulus regulation of localization regulation of immune system process lipid localization hexose metabolic process amine metabolic process reproductive process steroid metabolic process anatomical structure morphogenesis antigen processing and presentation cellular response to stress glycoprotein biosynthetic process lipid catabolic process cellular component biogenesis positive regulation of immune system process fatty acid biosynthetic process vesicle-mediated transport response to wounding cell division positive regulation of cellular metabolic process cellular amino acid and derivative metabolic process

Probe Genes 11 11 11 11 11 11 11 11 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7

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S4 Table S1. Cont.

Name [GO:0010033] [GO:0051128] [GO:0006163] [GO:0051641] [GO:0051276] [GO:0051173] [GO:0006325] [GO:0002526] [GO:0045454] [GO:0046907] [GO:0070887] [GO:0050776] [GO:0051707] [GO:0032535] [GO:0002252] [GO:0051605] [GO:0043069] [GO:0006820] [GO:0051239] [GO:0009260] [GO:0016070] [GO:0065009] [GO:0006869] [GO:0007167] [GO:0008643] [GO:0016051] [GO:0016052] [GO:0042127] [GO:0019953] [GO:0006644] [GO:0040008] [GO:0055085] [GO:0007399] [GO:0050790] [GO:0050793] [GO:0022900] [GO:0016568] [GO:0007243] [GO:0009888] [GO:0048878] [GO:0050778] [GO:0010604] [GO:0044106] [GO:0006357]

Describtion response to organic substance regulation of cellular component organization purine nucleotide metabolic process cellular localization chromosome organization positive regulation of nitrogen compound metabolic process chromatin organization acute inflammatory response cell redox homeostasis intracellular transport cellular response to chemical stimulus regulation of immune response response to other organism regulation of cellular component size immune effector process protein maturation by peptide bond cleavage negative regulation of programmed cell death anion transport regulation of multicellular organismal process ribonucleotide biosynthetic process cellular RNA metabolic process regulation of molecular function lipid transport enzyme linked receptor protein signaling pathway carbohydrate transport carbohydrate biosynthetic process carbohydrate catabolic process regulation of cell proliferation sexual reproduction phospholipid metabolic process regulation of growth transmembrane transport nervous system development regulation of catalytic activity regulation of developmental process electron transport chain chromatin modification protein kinase cascade tissue development chemical homeostasis positive regulation of immune response positive regulation of macromolecule metabolic process cellular amine metabolic process regulation of transcription from RNA polymerase II promoter

Probe Genes 7 7 7 7 7 7 7 7 7 7 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

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S5 Table S1. Cont.

Name [GO:0006259] [GO:0016125] [GO:0031328] [GO:0006090] [GO:0007010] [GO:0006814] [GO:0009199] [GO:0051129] [GO:0006694] [GO:0006886] [GO:0051246] [GO:0016071] [GO:0065003] [GO:0006119] [GO:0048646] [GO:0009108] [GO:0002520] [GO:0034984] [GO:0001501] [GO:0009628] [GO:0033043] [GO:0040007] [GO:0044087] [GO:0002504] [GO:0008361] [GO:0006511] [GO:0006800] [GO:0048609] [GO:0010605] [GO:0008380] [GO:0006725] [GO:0051049] [GO:0000087] [GO:0008203] [GO:0006520] [GO:0006916] [GO:0016126] [GO:0006094] [GO:0030155] [GO:0006096] [GO:0006457] [GO:0030258] [GO:0071310]

Describtion cellular DNA metabolic process sterol metabolic process positive regulation of cellular biosynthetic process pyruvate metabolic process cytoskeleton organization sodium ion transport ribonucleoside triphosphate metabolic process negative regulation of cellular component organization steroid biosynthetic process intracellular protein transport regulation of protein metabolic process mRNA metabolic process macromolecular complex assembly oxidative phosphorylation anatomical structure formation involved in morphogenesis coenzyme biosynthetic process immune system development cellular response to DNA damage stimulus skeletal system development response to abiotic stimulus regulation of organelle organization growth regulation of cellular component biogenesis antigen processing and presentation of peptide or polysaccharide antigen via MHC class II regulation of cell size ubiquitin-dependent protein catabolic process oxygen and reactive oxygen species metabolic process reproductive process in a multicellular organism negative regulation of macromolecule metabolic process RNA splicing cellular aromatic compound metabolic process regulation of transport M phase of mitotic cell cycle cholesterol metabolic process cellular amino acid metabolic process anti-apoptosis sterol biosynthetic process gluconeogenesis regulation of cell adhesion glycolysis protein folding lipid modification cellular response to organic substance

Probe Genes 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

4

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

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S6 Table S1. Cont.

Name [GO:0051336] [GO:0080134] [GO:0006575] [GO:0006818] [GO:0051726] [GO:0061024] [GO:0061020] [GO:0015674] [GO:0046395] [GO:0008284] [GO:0034621] [GO:0010646] [GO:0006650] [GO:0001503] [GO:0016049] [GO:0009719] [GO:0048872] [GO:0009617] [GO:0045137] [GO:0009615] [GO:0055082] [GO:0009116] [GO:0006801] [GO:0007610] [GO:0045884] [GO:0031347] [GO:0031333] [GO:0007259] [GO:0031324] [GO:0032989] [GO:0005976] [GO:0043281] [GO:0048511] [GO:0009062] [GO:0006816] [GO:0007017] [GO:0001568] [GO:0007346] [GO:0042325] [GO:0006790] [GO:0006695] [GO:0019221] [GO:0001558]

Describtion regulation of hydrolase activity regulation of response to stress cellular amino acid derivative metabolic process hydrogen transport regulation of cell cycle membrane organization positive regulation of transcription di-, tri-valent inorganic cation transport carboxylic acid catabolic process positive regulation of cell proliferation cellular macromolecular complex subunit organization regulation of cell communication glycerophospholipid metabolic process ossification cell growth response to endogenous stimulus homeostasis of number of cells response to bacterium development of primary sexual characteristics response to virus cellular chemical homeostasis nucleoside metabolic process superoxide metabolic process behavior regulation of survival gene product expression regulation of defense response negative regulation of protein complex assembly JAK-STAT cascade negative regulation of cellular metabolic process cellular component morphogenesis polysaccharide metabolic process regulation of caspase activity rhythmic process fatty acid catabolic process calcium ion transport microtubule-based process blood vessel development regulation of mitotic cell cycle regulation of phosphorylation sulfur metabolic process cholesterol biosynthetic process cytokine-mediated signaling pathway regulation of cell growth

Probe Genes 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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S7 Table S1. Cont.

Name [GO:0007178] [GO:0006979] [GO:0045927] [GO:0015985] [GO:0019216] [GO:0006897] [GO:0007154] [GO:0006470] [GO:0045944] [GO:0006461] [GO:0007169] [GO:0051270]

Describtion transmembrane receptor protein serine/threonine kinase signaling pathway response to oxidative stress positive regulation of growth energy coupled proton transport, down electrochemical gradient regulation of lipid metabolic process endocytosis cell communication protein amino acid dephosphorylation positive regulation of transcription from RNA polymerase II promoter protein complex assembly transmembrane receptor protein tyrosine kinase signaling pathway regulation of cellular component movement

Probe Genes 3

3

3 3 3 3 3 3 3

3 3 3 3 3 3 3

3

3

3 3 3

3 3 3

Table S2. The significant gene ontology cellular components in swines. Name [GO:0044422] [GO:0005634] [GO:0044446] [GO:0032991] [GO:0005886] [GO:0005576] [GO:0043234] [GO:0005739] [GO:0005783] [GO:0043228] [GO:0044459] [GO:0031090] [GO:0005794] [GO:0044429] [GO:0044421] [GO:0031974] [GO:0005856] [GO:0012505] [GO:0031975] [GO:0031982] [GO:0044430] [GO:0044428] [GO:0016023] [GO:0005740] [GO:0005829] [GO:0031966] [GO:0071212] [GO:0005759]

Description organelle part nucleus intracellular organelle part macromolecular complex plasma membrane extracellular region protein complex mitochondrion endoplasmic reticulum non-membrane-bounded organelle plasma membrane part organelle membrane Golgi apparatus mitochondrial part extracellular region part membrane-enclosed lumen cytoskeleton endomembrane system envelope vesicle cytoskeletal part nuclear part cytoplasmic membrane-bounded vesicle mitochondrial envelope cytosol mitochondrial membrane subsynaptic reticulum mitochondrial matrix

Probe 69 68 68 62 60 52 49 36 34 32 30 30 28 23 22 21 20 17 17 15 14 14 13 13 12 12 11 11

Genes 69 68 68 62 60 52 49 36 34 32 30 30 28 23 22 21 20 17 17 15 14 14 13 13 12 12 11 11

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S8 Table S2. Cont.

Name [GO:0019898] [GO:0015630] [GO:0031012] [GO:0030529] [GO:0005773] [GO:0005578] [GO:0009898] [GO:0005792] [GO:0048770] [GO:0005764] [GO:0044432] [GO:0044431] [GO:0031981] [GO:0042611] [GO:0005840] [GO:0000502] [GO:0005743] [GO:0000139] [GO:0005789] [GO:0031225] [GO:0005654] [GO:0005874] [GO:0005819] [GO:0042613] [GO:0031968] [GO:0044451] [GO:0031300] [GO:0005887] [GO:0005635] [GO:0005911] [GO:0005777] [GO:0005768] [GO:0046930] [GO:0015629] [GO:0016469] [GO:0005694] [GO:0031301] [GO:0048471] [GO:0043292] [GO:0045202] [GO:0031965] [GO:0016323] [GO:0005681] [GO:0033176] [GO:0005730] [GO:0016604] [GO:0022624]

Description extrinsic to membrane microtubule cytoskeleton extracellular matrix ribonucleoprotein complex vacuole proteinaceous extracellular matrix internal side of plasma membrane microsome pigment granule lysosome endoplasmic reticulum part Golgi apparatus part nuclear lumen MHC protein complex ribosome proteasome complex mitochondrial inner membrane Golgi membrane endoplasmic reticulum membrane anchored to membrane nucleoplasm microtubule spindle MHC class II protein complex organelle outer membrane nucleoplasm part intrinsic to organelle membrane integral to plasma membrane nuclear envelope cell-cell junction peroxisome endosome pore complex actin cytoskeleton proton-transporting two-sector ATPase complex chromosome integral to organelle membrane perinuclear region of cytoplasm contractile fiber synapse nuclear membrane basolateral plasma membrane spliceosomal complex proton-transporting V-type ATPase complex nucleolus nuclear body proteasome accessory complex

Probe 10 10 10 10 9 9 9 8 8 8 8 8 8 7 7 7 7 7 6 6 6 6 5 5 5 5 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3

Genes 10 10 10 10 9 9 9 8 8 8 8 8 8 7 7 7 7 7 6 6 6 6 5 5 5 5 4 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 3 3 3 3

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S9

Table S3. The significant gene ontology molecular functions in swines. Name [GO:0000166] [GO:0017076] [GO:0016787] [GO:0046914] [GO:0032555] [GO:0016740] [GO:0030554] [GO:0003676] [GO:0032559] [GO:0005524] [GO:0016491] [GO:0005215] [GO:0060089] [GO:0008270] [GO:0022892] [GO:0004872] [GO:0022857] [GO:0030528] [GO:0016772] [GO:0003677] [GO:0022891] [GO:0016301] [GO:0015075] [GO:0008233] [GO:0016773] [GO:0005102] [GO:0070011] [GO:0048037] [GO:0003700] [GO:0004672] [GO:0004175] [GO:0008324] [GO:0005509] [GO:0005506] [GO:0050662] [GO:0030234] [GO:0019001] [GO:0016788] [GO:0009055] [GO:0004888] [GO:0016817] [GO:0005198] [GO:0004857] [GO:0004674] [GO:0022890]

Describtion nucleotide binding purine nucleotide binding hydrolase activity transition metal ion binding purine ribonucleotide binding transferase activity adenyl nucleotide binding nucleic acid binding adenyl ribonucleotide binding ATP binding oxidoreductase activity transporter activity molecular transducer activity zinc ion binding substrate-specific transporter activity receptor activity transmembrane transporter activity transcription regulator activity transferase activity, transferring phosphorus-containing groups DNA binding substrate-specific transmembrane transporter activity kinase activity ion transmembrane transporter activity peptidase activity phosphotransferase activity, alcohol group as acceptor receptor binding peptidase activity, acting on L-amino acid peptides cofactor binding transcription factor activity protein kinase activity endopeptidase activity cation transmembrane transporter activity calcium ion binding iron ion binding coenzyme binding enzyme regulator activity guanyl nucleotide binding hydrolase activity, acting on ester bonds electron carrier activity transmembrane receptor activity hydrolase activity, acting on acid anhydrides structural molecule activity enzyme inhibitor activity protein serine/threonine kinase activity inorganic cation transmembrane transporter activity

Probe 96 78 73 70 70 68 61 56 53 52 50 50 49 43 39 37 35 34 34 34 33 30 30 29 27 26 25 25 25 24 22 21 21 21 20 19 18 16 16 16 15 15 15 15 14

Genes 96 78 73 70 70 68 61 56 53 52 50 50 49 43 39 37 35 34 34 34 33 30 30 29 27 26 25 25 25 24 22 21 21 21 20 19 18 16 16 16 15 15 15 15 14

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S10 Table S3. Cont.

Name [GO:0000287] [GO:0022804] [GO:0008092] [GO:0030414] [GO:0016757] [GO:0043565] [GO:0017171] [GO:0016746] [GO:0016614] [GO:0016874] [GO:0016705] [GO:0046906] [GO:0016887] [GO:0008289] [GO:0008234] [GO:0004930] [GO:0050660] [GO:0046873] [GO:0042623] [GO:0042578] [GO:0022838] [GO:0020037] [GO:0019842] [GO:0016747] [GO:0015078] [GO:0005125] [GO:0004197] [GO:0003723] [GO:0051287] [GO:0046983] [GO:0030246] [GO:0016791] [GO:0016758] [GO:0015291] [GO:0008083] [GO:0005126] [GO:0004713] [GO:0003779] [GO:0003735] [GO:0031406] [GO:0030145] [GO:0022836] [GO:0016879]

Describtion magnesium ion binding active transmembrane transporter activity cytoskeletal protein binding peptidase inhibitor activity transferase activity, transferring glycosyl groups sequence-specific DNA binding serine hydrolase activity transferase activity, transferring acyl groups oxidoreductase activity, acting on CH-OH group of donors ligase activity oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen tetrapyrrole binding ATPase activity lipid binding cysteine-type peptidase activity G-protein coupled receptor activity FAD binding metal ion transmembrane transporter activity ATPase activity, coupled phosphoric ester hydrolase activity substrate-specific channel activity heme binding vitamin binding transferase activity, transferring acyl groups other than amino-acyl groups hydrogen ion transmembrane transporter activity cytokine activity cysteine-type endopeptidase activity RNA binding NAD or NADH binding protein dimerization activity carbohydrate binding phosphatase activity transferase activity, transferring hexosyl groups secondary active transmembrane transporter activity growth factor activity cytokine receptor binding protein tyrosine kinase activity actin binding structural constituent of ribosome carboxylic acid binding manganese ion binding gated channel activity ligase activity, forming carbon-nitrogen bonds

Probe 14 13 13 12 12 11 11 11 11 10

Genes 14 13 13 12 12 11 11 11 11 10

10

10

9 9 9 9 9 8 8 8 8 8 8 8

9 9 9 9 9 8 8 8 8 8 8 8

8

8

8 8 8 8 7 7 7 7 7 7 7 7 7 7 7 6 6 6 6

8 8 8 8 7 7 7 7 7 7 7 7 7 7 7 6 6 6 6

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S11 Table S3. Cont.

Name [GO:0016810] [GO:0016209] [GO:0008509] [GO:0008430] [GO:0004721] [GO:0004497] [GO:0050661] [GO:0042626] [GO:0031420] [GO:0019904] [GO:0016853] [GO:0016811] [GO:0016798] [GO:0016779] [GO:0016563] [GO:0015293] [GO:0015082] [GO:0005507] [GO:0004867] [GO:0070403] [GO:0070279] [GO:0051540] [GO:0046915] [GO:0042625] [GO:0019955] [GO:0019899] [GO:0019199] [GO:0016881] [GO:0016860] [GO:0016741] [GO:0016709] [GO:0016684] [GO:0016627] [GO:0015631] [GO:0008134] [GO:0005529] [GO:0005516] [GO:0005253] [GO:0004869] [GO:0004386] [GO:0004091] [GO:0001664]

Describtion hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds antioxidant activity anion transmembrane transporter activity selenium binding phosphoprotein phosphatase activity monooxygenase activity NADP or NADPH binding ATPase activity, coupled to transmembrane movement of substances alkali metal ion binding protein domain specific binding isomerase activity hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides hydrolase activity, acting on glycosyl bonds nucleotidyltransferase activity transcription activator activity symporter activity di-, tri-valent inorganic cation transmembrane transporter activity copper ion binding serine-type endopeptidase inhibitor activity NAD binding vitamin B6 binding metal cluster binding transition metal ion transmembrane transporter activity ATPase activity, coupled to transmembrane movement of ions cytokine binding enzyme binding transmembrane receptor protein kinase activity acid-amino acid ligase activity intramolecular oxidoreductase activity transferase activity, transferring one-carbon groups oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NADH or NADPH as one donor, and incorporation of one atom of oxygen oxidoreductase activity, acting on peroxide as acceptor oxidoreductase activity, acting on the CH-CH group of donors tubulin binding transcription factor binding sugar binding calmodulin binding anion channel activity cysteine-type endopeptidase inhibitor activity helicase activity carboxylesterase activity G-protein-coupled receptor binding

Probe 6 6 6 6 6 6 5 5 5 5 5

Genes 6 6 6 6 6 6 5 5 5 5 5

5

5

5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4

5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4

4

4

4 4 4 4 4 4 4 4 4 4 4

4 4 4 4 4 4 4 4 4 4 4

Int. J. Mol. Sci. 2013, 14

S12 Table S3. Cont.

Name [GO:0060589] [GO:0051427] [GO:0046943] [GO:0046912] [GO:0042803] [GO:0031402] [GO:0022843] [GO:0019838] [GO:0019829] [GO:0019787] [GO:0016829] [GO:0016790] [GO:0016769] [GO:0016712] [GO:0016702] [GO:0016675] [GO:0016667] [GO:0016651] [GO:0016407] [GO:0016298] [GO:0015929] [GO:0015294] [GO:0015144] [GO:0010860] [GO:0008565] [GO:0008528] [GO:0008308] [GO:0008170] [GO:0008026] [GO:0008009] [GO:0005539] [GO:0005385] [GO:0005179] [GO:0004879] [GO:0004725] [GO:0004715] [GO:0004576] [GO:0004198] [GO:0003995] [GO:0003743] [GO:0003712]

Describtion nucleoside-triphosphatase regulator activity hormone receptor binding carboxylic acid transmembrane transporter activity transferase activity, transferring acyl groups, acyl groups converted into alkyl on transfer protein homodimerization activity sodium ion binding voltage-gated cation channel activity growth factor binding cation-transporting ATPase activity small conjugating protein ligase activity lyase activity thiolester hydrolase activity transferase activity, transferring nitrogenous groups oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, reduced flavin or flavoprotein as one donor, and incorporation of one atom of oxygen oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen oxidoreductase activity, acting on heme group of donors oxidoreductase activity, acting on sulfur group of donors oxidoreductase activity, acting on NADH or NADPH acetyltransferase activity lipase activity hexosaminidase activity solute:cation symporter activity carbohydrate transmembrane transporter activity proteasome regulator activity protein transporter activity peptide receptor activity, G-protein coupled voltage-gated anion channel activity N-methyltransferase activity ATP-dependent helicase activity chemokine activity glycosaminoglycan binding zinc ion transmembrane transporter activity hormone activity ligand-dependent nuclear receptor activity protein tyrosine phosphatase activity non-membrane spanning protein tyrosine kinase activity oligosaccharyl transferase activity calcium-dependent cysteine-type endopeptidase activity acyl-CoA dehydrogenase activity translation initiation factor activity transcription cofactor activity

Probe 3 3 3

Genes 3 3 3

3

3

3 3 3 3 3 3 3 3 3

3 3 3 3 3 3 3 3 3

3

3

3

3

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Int. J. Mol. Sci. 2013, 14

S13 Table S4. Pathways enriched in phenotype TG.

Pathway ssc04370:VEGF signaling pathway ssc04722:Neurotrophin signaling pathway ssc04610:Complement and coagulation cascades ssc04660:T cell receptor signaling pathway ssc00410:beta-Alanine metabolism ssc05216:Thyroid cancer ssc04810:Regulation of actin cytoskeleton ssc04012:ErbB signaling pathway ssc04330:Notch signaling pathway ssc04080:Neuroactive ligand-receptor interaction ssc00520:Amino sugar and nucleotide sugar metabolism ssc04730:Long-term depression ssc00640:Propanoate metabolism ssc04664:Fc epsilon RI signaling pathway ssc04912:GnRH signaling pathway ssc04662:B cell receptor signaling pathway ssc00650:Butanoate metabolism ssc04621:NOD-like receptor signaling pathway ssc00310:Lysine degradation ssc05211:Renal cell carcinoma ssc05221:Acute myeloid leukemia

Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank At Max

6

−0.76

−1.62

0.009

1

0.774

46

5

−0.74

−1.54

0.029

1

0.95

49

9

−0.61

−1.5

0.054

1

0.981

24

14

−0.53

−1.46

0.072

1

0.995

134

5

−0.68

−1.4

0.09

1

1

22

6

−0.63

−1.39

0.118

1

1

35

16

−0.48

−1.38

0.123

1

1

48

16

−0.49

−1.37

0.105

1

1

17

5

−0.67

−1.37

0.108

1

1

61

9

−0.55

−1.36

0.133

1

1

20

5

−0.65

−1.34

0.124

1

1

60

18

−0.45

−1.34

0.132

1

1

77

9

−0.54

−1.32

0.155

1

1

42

8

−0.56

−1.31

0.144

1

1

132

6

−0.61

−1.31

0.148

1

1

49

12

−0.48

−1.3

0.18

1

1

134

6

−0.6

−1.29

0.181

1

1

121

12

−0.49

−1.29

0.166

0.95

1

145

6

−0.59

−1.28

0.168

0.945

1

122

8

−0.54

−1.27

0.188

0.927

1

132

14

−0.47

−1.27

0.175

0.904

1

132

Leading Edge tags = 67%, list = 13%, signal = 76% tags = 60%, list = 14%, signal = 69% tags = 44%, list = 7%, signal = 47% tags = 79%, list = 39%, signal = 123% tags = 40%, list = 6%, signal = 42% tags = 33%, list = 10%, signal = 36% tags = 31%, list = 14%, signal = 35% tags = 19%, list = 5%, signal = 19% tags = 60%, list = 18%, signal = 72% tags = 33%, list = 6%, signal = 34% tags = 60%, list = 17%, signal = 72% tags = 44%, list = 22%, signal = 54% tags = 33%, list = 12%, signal = 37% tags = 75%, list = 38%, signal = 119% tags = 50%, list = 14%, signal = 57% tags = 67%, list = 39%, signal = 105% tags = 83%, list = 35%, signal = 126% tags = 75%, list = 42%, signal = 125% tags = 83%, list = 35%, signal = 127% tags = 75%, list = 38%, signal = 119% tags = 71%, list = 38%, signal = 111%

Int. J. Mol. Sci. 2013, 14

S14 Table S4. Cont. Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank At Max

16

−0.45

−1.27

0.207

0.874

1

113

3

−0.71

−1.24

0.197

0.927

1

49

2

−0.77

−1.22

0.218

0.974

1

8

15

−0.44

−1.21

0.223

0.975

1

105

19

−0.4

−1.21

0.244

0.94

1

52

2

−0.76

−1.21

0.193

0.917

1

85

7

−0.54

−1.21

0.24

0.885

1

27

13

−0.43

−1.19

0.251

0.939

1

164

ssc03050:Proteasome

7

−0.51

−1.18

0.283

0.92

1

82

ssc04114:Oocyte meiosis

9

−0.49

−1.18

0.28

0.902

1

49

7

−0.52

−1.18

0.291

0.883

1

78

12

−0.44

−1.18

0.279

0.859

1

121

7

−0.52

−1.17

0.28

0.847

1

46

3

−0.66

−1.17

0.266

0.827

1

121

9

−0.47

−1.17

0.293

0.816

1

188

4

−0.61

−1.16

0.272

0.803

1

56

2

−0.73

−1.16

0.267

0.786

1

49

5

−0.57

−1.16

0.313

0.778

1

114

5

−0.55

−1.16

0.312

0.76

1

8

5

−0.55

−1.15

0.29

0.768

1

26

Pathway ssc05212:Pancreatic cancer ssc04270:Vascular smooth muscle contraction ssc00053:Ascorbate and aldarate metabolism ssc05222:Small cell lung cancer ssc04020:Calcium signaling pathway ssc00260:Glycine, serine and threonine metabolism ssc00620:Pyruvate metabolism ssc04130:SNARE interactions in vesicular transport

ssc04640:Hematopoietic cell lineage ssc00280:Valine, leucine and isoleucine degradation ssc04914:Progesterone-medi ated oocyte maturation ssc00903:Limonene and pinene degradation ssc00071:Fatty acid metabolism ssc00380:Tryptophan metabolism ssc04740:Olfactory transduction ssc00564:Glycerophospholi pid metabolism ssc00561:Glycerolipid metabolism ssc00980:Metabolism of xenobiotics by cytochrome P450

Leading Edge tags = 63%, list = 33%, signal = 89% tags = 67%, list = 14%, signal = 77% tags = 50%, list = 2%, signal = 51% tags = 53%, list = 31%, signal = 73% tags = 26%, list = 15%, signal = 29% tags = 100%, list = 25%, signal = 132% tags = 29%, list = 8%, signal = 30% tags = 77%, list = 48%, signal = 141% tags = 43%, list = 24%, signal = 55% tags = 33%, list = 14%, signal = 38% tags = 57%, list = 23%, signal = 72% tags = 58%, list = 35%, signal = 87% tags = 43%, list = 13%, signal = 48% tags = 100%, list = 35%, signal = 153% tags = 100%, list = 55%, signal = 215% tags = 50%, list = 16%, signal = 59% tags = 50%, list = 14%, signal = 58% tags = 80%, list = 33%, signal = 118% tags = 20%, list = 2%, signal = 20% tags = 40%, list = 8%, signal = 43%

Int. J. Mol. Sci. 2013, 14

S15 Table S4. Cont. Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank At Max

8

−0.48

−1.14

0.309

0.759

1

134

15

−0.4

−1.13

0.325

0.777

1

80

4

−0.59

−1.13

0.322

0.762

1

93

6

−0.51

−1.12

0.326

0.759

1

49

4

−0.58

−1.12

0.337

0.761

1

41

14

−0.4

−1.12

0.339

0.748

1

132

2

−0.71

−1.11

0.346

0.741

1

16

2

−0.7

−1.1

0.346

0.746

1

107

8

−0.48

−1.1

0.368

0.749

1

109

9

−0.45

−1.09

0.385

0.744

1

67

11

−0.43

−1.09

0.363

0.745

1

94

10

−0.43

−1.08

0.385

0.744

1

23

2

−0.67

−1.08

0.367

0.737

1

116

3

−0.6

−1.08

0.379

0.727

1

139

20

−0.34

−1.03

0.445

0.811

1

184

2

−0.65

−1.03

0.442

0.803

1

124

7

−0.44

−1.02

0.441

0.804

1

131

5

−0.5

−1.01

0.446

0.814

1

93

ssc04144:Endocytosis

13

−0.37

−1

0.491

0.831

1

19

ssc05020:Prion diseases

5

−0.49

−1

0.49

0.822

1

25

ssc04540:Gap junction

8

−0.42

−1

0.48

0.81

1

20

Pathway ssc04916:Melanogenesis ssc04630:Jak-STAT signaling pathway ssc00350:Tyrosine metabolism ssc04070:Phosphatidylinosit ol signaling system ssc00603:Glycosphingolipid biosynthesis ssc04650:Natural killer cell mediated cytotoxicity ssc00140:Steroid hormone biosynthesis ssc00600:Sphingolipid metabolism ssc03040:Spliceosome ssc00010:Glycolysis/ Gluconeogenesis ssc00190:Oxidative phosphorylation ssc04530:Tight junction ssc01040:Biosynthesis of unsaturated fatty acids ssc00450:Selenoamino acid metabolism ssc04210:Apoptosis ssc00900:Terpenoid backbone biosynthesis ssc00330:Arginine and proline metabolism ssc00360:Phenylalanine metabolism

Leading Edge tags = 63%, list = 39%, signal = 100% tags = 53%, list = 23%, signal = 66% tags = 75%, list = 27%, signal = 102% tags = 50%, list = 14%, signal = 57% tags = 50%, list = 12%, signal = 56% tags = 57%, list = 38%, signal = 89% tags = 50%, list = 5%, signal = 52% tags = 100%, list = 31%, signal = 144% tags = 50%, list = 32%, signal = 71% tags = 33%, list = 19%, signal = 40% tags = 36%, list = 27%, signal = 48% tags = 20%, list = 7%, signal = 21% tags = 100%, list = 34%, signal = 150% tags = 100%, list = 40%, signal = 166% tags = 70%, list = 53%, signal = 142% tags = 100%, list = 36%, signal = 155% tags = 71%, list = 38%, signal = 113% tags = 60%, list = 27%, signal = 81% tags = 15%, list = 6%, signal = 16% tags = 40%, list = 7%, signal = 43% tags = 25%, list = 6%, signal = 26%

Int. J. Mol. Sci. 2013, 14

S16 Table S4. Cont. Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank At Max

ssc00020:Citrate cycle (TCA cycle)

11

−0.37

−0.97

0.502

0.858

1

221

ssc05214:Glioma

14

−0.35

−0.97

0.513

0.845

1

49

9

−0.4

−0.97

0.522

0.834

1

45

2

−0.61

−0.97

0.538

0.823

1

135

13

−0.35

−0.96

0.509

0.814

1

134

4

−0.51

−0.96

0.544

0.818

1

60

2

−0.61

−0.95

0.568

0.81

1

135

7

−0.43

−0.95

0.549

0.799

1

148

9

−0.38

−0.94

0.542

0.808

1

105

3

−0.55

−0.94

0.561

0.801

1

159

15

−0.34

−0.94

0.538

0.792

1

140

4

−0.48

−0.93

0.569

0.802

1

56

2

−0.6

−0.93

0.583

0.791

1

16

12

−0.35

−0.93

0.565

0.785

1

45

8

−0.39

−0.92

0.575

0.79

1

16

3

−0.52

−0.92

0.608

0.782

1

38

3

−0.52

−0.91

0.604

0.786

1

135

10

−0.36

−0.9

0.576

0.789

1

186

15

−0.32

−0.89

0.615

0.789

1

64

18

−0.3

−0.88

0.644

0.807

1

134

6

−0.4

−0.86

0.648

0.824

1

45

Pathway

ssc05223:Non-small cell lung cancer ssc00604:Glycosphingolipid biosynthesis ssc05210:Colorectal cancer ssc00500:Starch and sucrose metabolism ssc00511:Other glycan degradation ssc04360:Axon guidance ssc04623:Cytosolic DNA-sensing pathway ssc00040:Pentose and glucuronate interconversions ssc04920:Adipocytokine signaling pathway ssc00340:Histidine metabolism ssc00591:Linoleic acid metabolism ssc05213:Endometrial cancer ssc00982:Drug metabolism ssc00830:Retinol metabolism ssc00531:Glycosaminoglyca n degradation ssc05014:Amyotrophic lateral sclerosis (ALS) ssc04310:Wnt signaling pathway ssc04062:Chemokine signaling pathway ssc04150:mTOR signaling pathway

Leading Edge tags = 100%, list = 64%, signal = 271% tags = 29%, list = 14%, signal = 32% tags = 33%, list = 13%, signal = 37% tags = 100%, list = 39%, signal = 164% tags = 54%, list = 39%, signal = 85% tags = 50%, list = 17%, signal = 60% tags = 100%, list = 39%, signal = 164% tags = 71%, list = 43%, signal = 123% tags = 44%, list = 31%, signal = 62% tags = 100%, list = 46%, signal = 184% tags = 73%, list = 41%, signal = 118% tags = 50%, list = 16%, signal = 59% tags = 50%, list = 5%, signal = 52% tags = 25%, list = 13%, signal = 28% tags = 25%, list = 5%, signal = 26% tags = 67%, list = 11%, signal = 74% tags = 67%, list = 39%, signal = 109% tags = 80%, list = 54%, signal = 169% tags = 27%, list = 19%, signal = 31% tags = 61%, list = 39%, signal = 95% tags = 33%, list = 13%, signal = 38%

Int. J. Mol. Sci. 2013, 14

S17 Table S4. Cont. Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank At Max

ssc04110:Cell cycle

14

−0.31

−0.86

0.643

0.817

1

138

ssc00270:Cysteine and methionine metabolism

5

−0.42

−0.85

0.666

0.816

1

171

ssc05218:Melanoma

11

−0.32

−0.83

0.689

0.845

1

45

17

−0.28

−0.83

0.701

0.835

1

65

10

−0.33

−0.82

0.69

0.835

1

31

11

−0.32

−0.82

0.702

0.829

1

119

3

−0.46

−0.82

0.726

0.821

1

159

8

−0.35

−0.81

0.718

0.823

1

35

2

−0.51

−0.8

0.776

0.828

1

171

9

−0.31

−0.79

0.729

0.838

1

158

5

−0.38

−0.78

0.772

0.84

1

15

7

−0.33

−0.77

0.772

0.847

1

77

6

−0.35

−0.76

0.781

0.85

1

228

6

−0.35

−0.74

0.801

0.863

1

45

18

−0.24

−0.71

0.833

0.892

1

181

14

−0.25

−0.69

0.847

0.896

1

182

2

−0.44

−0.69

0.91

0.895

1

95

3

−0.36

−0.64

0.924

0.927

1

221

2

−0.4

−0.62

0.957

0.935

1

53

14

−0.22

−0.62

0.917

0.929

1

145

2

−0.31

−0.49

1

0.983

1

101

Pathway

ssc04620:Toll-like receptor signaling pathway ssc05332:Graft-versus-host disease ssc04670:Leukocyte transendothelial migration ssc00052:Galactose metabolism ssc05219:Bladder cancer ssc00400:Phenylalanine, tyrosine and tryptophan biosynthesis ssc04120:Ubiquitin mediated proteolysis ssc00590:Arachidonic acid metabolism ssc04666:Fc gamma R-mediated phagocytosis ssc00510:N-Glycan biosynthesis ssc04930:Type II diabetes mellitus ssc05016:HuntinTGon's disease ssc04115:p53 signaling pathway ssc00770:Pantothenate and CoA biosynthesis ssc00630:Glyoxylate and dicarboxylate metabolism ssc00030:Pentose phosphate pathway ssc04622:RIG-I-like receptor signaling pathway ssc00601:Glycosphingolipid biosynthesis

Leading Edge tags = 57%, list = 40%, signal = 92% tags = 80%, list = 50%, signal = 157% tags = 27%, list = 13%, signal = 30% tags = 29%, list = 19%, signal = 34% tags = 20%, list = 9%, signal = 21% tags = 45%, list = 35%, signal = 67% tags = 67%, list = 46%, signal = 123% tags = 25%, list = 10%, signal = 27% tags = 100%, list = 50%, signal = 198% tags = 56%, list = 46%, signal = 100% tags = 20%, list = 4%, signal = 21% tags = 29%, list = 22%, signal = 36% tags = 100%, list = 66%, signal = 291% tags = 33%, list = 13%, signal = 38% tags = 61%, list = 53%, signal = 122% tags = 71%, list = 53%, signal = 146% tags = 50%, list = 28%, signal = 69% tags = 100%, list = 64%, signal = 277% tags = 50%, list = 15%, signal = 59% tags = 57%, list = 42%, signal = 95% tags = 50%, list = 29%, signal = 70%

Int. J. Mol. Sci. 2013, 14

S18 Table S5. Pathways enriched in phenotype CG. Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank at Max

ssc03010:Ribosome

7

0.68

1.79

0.009

0.358

0.355

114

ssc05310:Asthma

7

0.66

1.73

0.027

0.284

0.521

68

ssc05330:Allograft rejection

11

0.54

1.7

0.025

0.221

0.575

80

10

0.52

1.6

0.036

0.307

0.784

80

6

0.63

1.6

0.039

0.25

0.791

53

9

0.53

1.53

0.074

0.309

0.908

80

12

0.43

1.45

0.084

0.42

0.978

61

9

0.48

1.41

0.108

0.451

0.994

68

ssc00230:Purine metabolism

4

0.66

1.39

0.094

0.434

0.995

28

ssc02010:ABC transporters

2

0.81

1.34

0.139

0.513

0.997

66

2

0.77

1.25

0.21

0.692

1

26

12

0.36

1.17

0.265

0.845

1

68

17

0.31

1.16

0.243

0.791

1

85

6

0.45

1.15

0.282

0.777

1

29

4

0.52

1.14

0.278

0.737

1

31

3

0.55

1.07

0.364

0.859

1

23

3

0.54

1.03

0.42

0.905

1

158

5

0.44

1.03

0.392

0.855

1

0

16

0.28

1.01

0.411

0.861

1

80

2

0.61

1

0.465

0.849

1

134

14

0.28

0.96

0.465

0.901

1

67

Pathways

ssc04940:Type I diabetes mellitus ssc04260:Cardiac muscle contraction ssc05320:Autoimmune thyroid disease ssc04060:Cytokine-cytokine receptor interaction ssc04672:Intestinal immune network for IgA production

ssc05412:Arrhythmogenic right ventricular cardiomyopathy (ARVC) ssc05322:Systemic lupus erythematosus ssc04612:Antigen processing and presentation ssc05414:Dilated cardiomyopathy ssc04340:Hedgehog signaling pathway ssc04512:ECM-receptor interaction ssc00240:Pyrimidine metabolism ssc04730:Long-term depression ssc04514:Cell adhesion molecules (CAMs) ssc00760:Nicotinate and nicotinamide metabolism ssc04350:TGF-beta signaling pathway

Leading Edge tags = 100%, list = 33%, signal = 147% tags = 86%, list = 20%, signal = 105% tags = 82%, list = 23%, signal = 103% tags = 80%, list = 23%, signal = 101% tags = 50%, list = 15%, signal = 58% tags = 89%, list = 23%, signal = 113% tags = 58%, list = 18%, signal = 68% tags = 78%, list = 20%, signal = 94% tags = 50%, list = 8%, signal = 54% tags = 100%, list = 19%, signal = 123% tags = 50%, list = 8%, signal = 54% tags = 58%, list = 20%, signal = 70% tags = 65%, list = 25%, signal = 82% tags = 33%, list = 8%, signal = 36% tags = 50%, list = 9%, signal = 54% tags = 67%, list = 7%, signal = 71% tags = 100%, list = 46%, signal = 183% tags = 20%, list = 0%, signal = 20% tags = 56%, list = 23%, signal = 70% tags = 100%, list = 39%, signal = 163% tags = 36%, list = 19%, signal = 43%

Int. J. Mol. Sci. 2013, 14

S19 Table S5. Cont. Size

ES

NES

NOM p-val

FDR q-val

FWER p-val

Rank at Max

12

0.28

0.94

0.528

0.904

1

251

4

0.44

0.94

0.513

0.878

1

8

5

0.39

0.93

0.541

0.864

1

16

2

0.55

0.91

0.564

0.859

1

156

2

0.54

0.9

0.583

0.86

1

14

2

0.53

0.87

0.633

0.877

1

162

ssc05217:Basal cell carcinoma

6

0.34

0.87

0.605

0.846

1

31

ssc00983:Drug metabolism

3

0.45

0.87

0.639

0.829

1

4

ssc05416:Viral myocarditis

15

0.24

0.84

0.636

0.842

1

80

ssc05410:Hypertrophic cardiomyopathy (HCM)

8

0.3

0.82

0.72

0.847

1

26

ssc04520:Adherens junction

10

0.27

0.82

0.675

0.826

1

44

3

0.42

0.8

0.722

0.833

1

13

8

0.28

0.8

0.731

0.812

1

18

3

0.41

0.79

0.766

0.81

1

61

13

0.22

0.77

0.769

0.81

1

25

2

0.38

0.6

0.973

0.97

1

213

4

0.26

0.57

0.955

0.962

1

32

Pathways ssc05012:Parkinson's disease ssc00562:Inositol phosphate metabolism ssc00250:Alanine, aspartate and glutamate metabolism ssc04130:SNARE interactions in vesicular transport ssc04140:Regulation of autophagy ssc00062:Fatty acid elongation in mitochondria

ssc04960:Aldosterone-regulate d sodium reabsorption ssc00480:Glutathione metabolism ssc05340:Primary immunodeficiency ssc03020:RNA polymerase ssc04320:Dorso-ventral axis formation ssc00051:Fructose and mannose metabolism

Leading Edge tags = 100%, list = 73%, signal = 357% tags = 25%, list = 2%, signal = 25% tags = 40%, list = 5%, signal = 41% tags = 100%, list = 45%, signal = 182% tags = 50%, list = 4%, signal = 52% tags = 100%, list = 47%, signal = 188% tags = 33%, list = 9%, signal = 36% tags = 33%, list = 1%, signal = 33% tags = 53%, list = 23%, signal = 66% tags = 25%, list = 8%, signal = 26% tags = 30%, list = 13%, signal = 33% tags = 33%, list = 4%, signal = 34% tags = 25%, list = 5%, signal = 26% tags = 67%, list = 18%, signal = 80% tags = 15%, list = 7%, signal = 16% tags = 100%, list = 62%, signal = 261% tags = 25%, list = 9%, signal = 27%

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