DOCSLIB.ORG

Table S2) Are Shown with the Promoter for CCL18 (Table 1 and Fig

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Table S2) Are Shown with the Promoter for CCL18 (Table 1 and Fig Supporting Information Canaan et al. 10.1073/pnas.0911676106 SI Text pooled scores from several arrays were averaged for each gene Tissue Culture. The human B cell lymphoma line, BJAB, and the and accepted if Ͼ2 for up-regulated genes or Ͻ0.5 for down- ϩ BJAB/EBNA1 cell lines, were kindly provided by the lab of Elliott regulated genes, because the scores of EBNA1 samples were Kieff (Harvard University). EBNA1 expression phenotype was superimposed on their cell line counterpart. verified by Western blot analysis before these studies and was reconfirmed by QRT-PCR while confirming microarray data. Quantitative Real-Time PCR (QRT-PCR). To verify and evaluate the Excluding the expression of EBNA1 gene, the cell lines in this study cDNA microarray data, samples were run using two independent are EBV-negative cell lines. The human epithelial cell line 293 and RNA preparations for BJAB and one RNA preparation for 293. the 293/EBNA1 were originated at the laboratory of Lawrence S. These samples were then reverse transcribed in triplicate, while Young (Institute for Cancer Studies, University of Birmingham each cDNA was analyzed in a single QRT-PCR. QRT-PCR Medical School, U.K.). reactions were run in ABI PRISM 7000 Sequence Detection System. The following oligo sequence pairs were used in the cDNA-Microarray Slides. The Peter MacCallum Cancer Institute QRT-PCR assays, sequences shown from 5Ј to 3Ј ends: Hs.833: (PMCI) microarrays used in this report contain approximately CAGCTCCATGTCGGTGTCAG and CCAATCTTCTGGGT- 10,500 cDNA clones representing 9,389 unique clones (UniGene GATCTGC; Hs.82396: CTATGCTTGGGAGCGAGGG and build 144) (25). CCCTGGGCTGTGTTGAAATG; Hs.62354: TACGATGG- GAAACTCTCTAGTGCC and AGCCCGTGGATTG- Purification of RNA, Amplification, Labeling, and Hybridization. Cell lines TACGTGA; Hs.75256: AATGTGCCAGTATGGCTCCC and were grown in four 750-mL tissue culture flasks (Falcon) to a GACACCCACAAAAGGCCAAG; Hs.77100: TCACA- density of 106 cells per 1 mL medium for BJAB and Ͻ70% GAAAAAGCGACGCTTT and CACTCCAGCCAAGTGT- confluence for the 293 under standard humidifying conditions. TCGTT; Hs.46423: AGACGCCGTCACCTATACGG and AT- RNA was purified in a two-step process involving extraction with an GGCTGTGACAGTTTTGCG; Hs.75450: AGCGTGG- organic solvent (TRIzol, Life Technologies) followed by column TGGCCATAGACA and CTTCACCAGATCCATGGCCT; chromatography (RNeasy, Qiagen). Microarray analysis was per- Hs.75573: GCCTTTGTCCAGAGGTGCAA and AGGACCTG- formed using two-color competitive hybridization of fluorescently GCTGAGAATCCAC; Hs.75703: CTCTCAGCACCAAT- labeled cDNA to the glass slide array, comparing EBNA1-positive GGGCTC and GTAAGAAAAGCAGCAGGCGG; Two pairs of cell line to its EBNA1-negative counterpart. RNA samples were oligos were used for MYC Hs.79070: CCGTCCAAGCAGAG- converted to cDNA, amplified, labeled, and hybridized as was GAGC and TCCGCAACAAGTCCTCTTCAG; CGAC- described in ref. 25. GAGAACAGTTGAAACACAA and CGCACAAGAGTTC- CGTAGCTG; EBNA1: CGACATCACACCATATACCGCA cDNA Microarray Scanning and Analysis. Slides were scanned using and ATTTCCAGGTCCTGTACCTGGC; the last pair was used to a dual UV-laser GSI Luminomics scanner (Hewlett Packard), confirm the presence or absence of EBNA1 in samples subjected to and fluorescence intensity was analyzed using the Quantarray QRT-PCR analysis. To overcome differences in initial mRNA program (Hewlett Packard). Images were formed by superim- amounts and to better quantify gene expression changes, we used posing the Cy3 and Cy5 images for each slide using the Scanalyze the mitogen-activated protein kinase 1 gene (MAPK1, Hs.324473) software (Eisen Lab). as a reference with the oligo pair: CCCTCGCATGACTGTTA- Scanning settings were used to equate intensity records of Cy3 CAGC and GGCACTTGGACAGTCATCTCTG. MAPK1 was and Cy5. Typically this means that Cy5 is scanned at 60% PMT not affected by EBNA1 expression by the microarray analysis. Oligo and Cy3 is scanned in 85% PMT. pairs with a Tm of 60 °C were chosen to generate 100 base pair PCR The numerical analysis of the scanned results was performed products. To verify and ensure QRT-PCR specificity, we screened in GeneSpring 5 (Silicon Genetics) and data were normalized for the human genome for each oligo pair using the ‘‘BLAST’’ (Basic Cy3/Cy5 ratio by applying LOWESS method to correct for Local Alignment Search Tool) protocol at the National Center for dye-specific intensity-dependant bias. Although this normaliza- Biotechnology Information (NCBI) web server. At the end of each tion produces more reliable results, it reduces the scores’ inten- QRT-PCR the synthesized products were verified for their speci- sity level. Data analysis of the normalized scores was carried out ficity by examining their actual Tm value. The sequence calculated by Access software (Microsoft) to pool results from several Tm for each PCR product was compared to the Tm measured experiments and then transferred to Excel software (Microsoft) through the decline of SYBR Green fluorescence from the melting for threshold cut off. To further reduce background signals, the strands in a gradual heat increase. Canaan et al. www.pnas.org/cgi/content/short/0911676106 1of5 Fig. S1. QR-PCR analysis and agarose gel fractionation for CHUK and MUC13 promoters show the formation of specific amplicons in ChIP DNA samples. Samples of BJAB (Ϫ) and BJAB/EBNA1 (ϩ) from three biological replicates, which were amplified by QR-PCR have demonstrated similar melting curves characteristics (Left). These samples were also fractionated on an agarose gel to further demonstrate amplicon specificity and amplification differences. QR-PCR for the reference gene beta Actin is shown for these biological replicates for BJAB/EBNA1 (ϩ) and its control BJAB (Ϫ). Canaan et al. www.pnas.org/cgi/content/short/0911676106 2of5 Fig. S2. Integrated genome browser view of ChIP-chip analysis for selected cellular promoters bound by EBNA1. The promoters for CHUK, MUC13, and OPN4 (Fig. 1, Fig. S1, and Table S2) are shown with the promoter for CCL18 (Table 1 and Fig. 1). The EBNA1 nonbound promoter for beta Actin (ACTB) is also shown as it was used as a reference for QR-PCR analysis and for the evaluation of enrichment folds. The EBNA1 nonbound PLAC9 and NRG3 promoter areas are also shown to further demonstrate selected promoter engagement by EBNA1. Canaan et al. www.pnas.org/cgi/content/short/0911676106 3of5 Table S1. QR-PCR analysis of selected genes affected by EBNA1 in BJAB and 293 cell lines according to the microarray data Unigen Official gene symbol and name BJAB EBNA1/BJAB 293 EBNA1/293 Hs.202453 MYC, V-myc avian myelocytomatosis viral oncogene homolog 0.173 Ϯ 0.0132 0.451 Ϯ 0.062 Hs.46423 H4FG, H4 histone family, member G 3.272 Ϯ 0.22 8.938 Ϯ 0.331 Hs.75703 CCL4, Chemokine (C-C motif) ligand 4 (homologous to mouse Mip-1b) 49.58 Ϯ 0.721 1.989 Ϯ 0.66 Hs.524760 OAS1, 2Ј,5Ј-oligoadenylate synthetase 1 (40–46 kDa) 0.052 Ϯ 0.042 0.503 Ϯ 0.08 Hs.458485 ISG15, Interferon-stimulated protein, 15 kDa 0.1 Ϯ 0.052 0.615 Ϯ 0.05 Hs.480938 LRBA, LPS-responsive vesicle trafficking, beach and anchor 0.231 Ϯ 0.062 0.043 Ϯ 0.08 Hs.75256 RGS1, Regulator of G-protein signalling 1 5.897 Ϯ 0.7581 1.21 Ϯ 0.27 Hs.77100 GTF2E2, General transcription factor IIE, polypeptide 2 (beta subunit, 34 kD) 4.777 Ϯ 1.01 0.558 Ϯ 0.05 Hs.75573 CENPE, Centromer protein E (312 kD) 0.575 Ϯ 0.112 2.049 Ϯ 0.01 Hs.522074 DSIPI, Delta sleep inducing peptide, immuno-reactor 0.817 Ϯ 0.02 2.412 Ϯ 0.221 Scores in table are given as a proportion of the curve slope values for EBNA1ϩ cell line over its cellular counterpart for the same PCR product. The slope was derived from the linear portion of the curve, which represents the fluorescent build up of the PCR products. Arrows indicate the score that initially was identified by microarray. Arrow direction represent up-regulation (1) and down-regulation (2) of the genes by microarray analysis. Dotted line separates the EBNA1-specific genes (above) from the tissue-specific genes (below). Scores are shown with their standard deviation that was generated from three QR-PCR samples. All samples were tested for EBNA1 expression by QR-PCR to verify EBNA1-positive and -negative samples. Canaan et al. www.pnas.org/cgi/content/short/0911676106 4of5 Table S2. Selected cellular promoters bound by EBNA1, ChIP-chip analysis # Chr Start End Signal Up-stream Annotation chr10 102295884 102296348 1.048 HIF1AN Hypoxia-inducible factor 1 alpha inhibitor chr11 88402235 88402812 1.021 OPN4* Opsin 4 chr10 102080666 102080996 1.014 PKD2L1 polycystic kidney disease 2-like 1 chr2 227897845 227897985 0.945 HRB Homo sapiens HIV-1 Rev binding protein (HRB) chr10 102035411 102035630 0.904 BLOC1S2 Biogenesis of lysosome-related organelles complex-1, subunit 2 chr10 102554161 102554629 0.888 PAX2 PAIRED BOX GENE 2 chr2 133896524 133896774 0.886 NAP5 Nck-associated protein 5 chr2 134753804 134753944 0.877 MGAT5 Mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase chr2 88705057 88705197 0.861 RPIA Ribose 5-phosphate isomerase A chr8 128815762 128816430 0.858 MYC V-myc avian myelocytomatosis viral oncogene homolog chr2 121946079 121946219 0.856 CLASP1 Cytoplasmic linker associated protein 1 chr2 37308770 37308910 0.852 PRKD3 Protein kinase D3 chr9 5175954 5176476 0.851 INSL6 Insulin like 6 chr9 97252681 97252853 0.833 FAM120A Hypothetical protein chr6 42303600 42303884 0.833 MRPS10 Mitochondrial ribosomal protein S10 chr9 129928476 129928648 0.806 LCN2 No annotations chr10 102083738 102084067 0.792 PKD2L1 polycystic kidney disease 2-like 1 chr10 68355365 68355588
Load more

Recommended publications
  • FK506-Binding Protein 12.6/1B, a Negative Regulator of [Ca2+], Rescues Memory and Restores Genomic Regulation in the Hippocampus of Aging Rats
    This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Neurobiology of Disease FK506-Binding Protein 12.6/1b, a negative regulator of [Ca2+], rescues memory and restores genomic regulation in the hippocampus of aging rats John C. Gant1, Eric M. Blalock1, Kuey-Chu Chen1, Inga Kadish2, Olivier Thibault1, Nada M. Porter1 and Philip W. Landfield1 1Department of Pharmacology & Nutritional Sciences, University of Kentucky, Lexington, KY 40536 2Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294 DOI: 10.1523/JNEUROSCI.2234-17.2017 Received: 7 August 2017 Revised: 10 October 2017 Accepted: 24 November 2017 Published: 18 December 2017 Author contributions: J.C.G. and P.W.L. designed research; J.C.G., E.M.B., K.-c.C., and I.K. performed research; J.C.G., E.M.B., K.-c.C., I.K., and P.W.L. analyzed data; J.C.G., E.M.B., O.T., N.M.P., and P.W.L. wrote the paper. Conflict of Interest: The authors declare no competing financial interests. NIH grants AG004542, AG033649, AG052050, AG037868 and McAlpine Foundation for Neuroscience Research Corresponding author: Philip W. Landfield, [email protected], Department of Pharmacology & Nutritional Sciences, University of Kentucky, 800 Rose Street, UKMC MS 307, Lexington, KY 40536 Cite as: J. Neurosci ; 10.1523/JNEUROSCI.2234-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published.
    [Show full text]
  • NIH Public Access Author Manuscript Pharmacogenomics
    NIH Public Access Author Manuscript Pharmacogenomics. Author manuscript; available in PMC 2014 June 12. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Pharmacogenomics. 2014 February ; 15(2): 137–146. doi:10.2217/pgs.13.213. Genome-wide Association and Pharmacological Profiling of 29 Anticancer Agents Using Lymphoblastoid Cell Lines Chad C. Brown1, Tammy M. Havener2, Marisa W. Medina3, John R. Jack1, Ronald M. Krauss3, Howard L. McLeod2, and Alison A. Motsinger-Reif1,2,* 1Bioinformatics Research Center, Department of Statistics, North Carolina State University, Raleigh, NC, 27607, USA 2Institute for Pharmacogenomics and Individualized Therapy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA 3Children’s Hospital Oakland Research Institute, Oakland, CA, 94609, USA Abstract Aims—Association mapping with lymphoblastoid cell lines (LCLs) is a promising approach in pharmacogenomics research, and in the current study we utilize this model to perform association mapping for 29 chemotherapy drugs. Materials and Methods—Currently, we use LCLs to perform genome-wide association mapping of the cytotoxic response of 520 European Americans to 29 different anticancer drugs, the largest LCL study to date. A novel association approach using a multivariate analysis of covariance design was employed with the software program MAGWAS, testing for differences in the dose-response profiles between genotypes without making assumptions about the response curve or the biological mode of association. Additionally, by classifying 25 of the 29 drugs into 8 families according to structural and mechanistic relationships, MAGWAS was used to test for associations that were shared across each drug family.
    [Show full text]
  • 1 Evidence for Gliadin Antibodies As Causative Agents in Schizophrenia
    1 Evidence for gliadin antibodies as causative agents in schizophrenia. C.J.Carter PolygenicPathways, 20 Upper Maze Hill, Saint-Leonard’s on Sea, East Sussex, TN37 0LG [email protected] Tel: 0044 (0)1424 422201 I have no fax Abstract Antibodies to gliadin, a component of gluten, have frequently been reported in schizophrenia patients, and in some cases remission has been noted following the instigation of a gluten free diet. Gliadin is a highly immunogenic protein, and B cell epitopes along its entire immunogenic length are homologous to the products of numerous proteins relevant to schizophrenia (p = 0.012 to 3e-25). These include members of the DISC1 interactome, of glutamate, dopamine and neuregulin signalling networks, and of pathways involved in plasticity, dendritic growth or myelination. Antibodies to gliadin are likely to cross react with these key proteins, as has already been observed with synapsin 1 and calreticulin. Gliadin may thus be a causative agent in schizophrenia, under certain genetic and immunological conditions, producing its effects via antibody mediated knockdown of multiple proteins relevant to the disease process. Because of such homology, an autoimmune response may be sustained by the human antigens that resemble gliadin itself, a scenario supported by many reports of immune activation both in the brain and in lymphocytes in schizophrenia. Gluten free diets and removal of such antibodies may be of therapeutic benefit in certain cases of schizophrenia. 2 Introduction A number of studies from China, Norway, and the USA have reported the presence of gliadin antibodies in schizophrenia 1-5. Gliadin is a component of gluten, intolerance to which is implicated in coeliac disease 6.
    [Show full text]
  • Detection of Interacting Transcription Factors in Human Tissues Using
    Myšičková and Vingron BMC Genomics 2012, 13(Suppl 1):S2 http://www.biomedcentral.com/1471-2164/13/S1/S2 PROCEEDINGS Open Access Detection of interacting transcription factors in human tissues using predicted DNA binding affinity Alena Myšičková*, Martin Vingron From The Tenth Asia Pacific Bioinformatics Conference (APBC 2012) Melbourne, Australia. 17-19 January 2012 Abstract Background: Tissue-specific gene expression is generally regulated by combinatorial interactions among transcription factors (TFs) which bind to the DNA. Despite this known fact, previous discoveries of the mechanism that controls gene expression usually consider only a single TF. Results: We provide a prediction of interacting TFs in 22 human tissues based on their DNA-binding affinity in promoter regions. We analyze all possible pairs of 130 vertebrate TFs from the JASPAR database. First, all human promoter regions are scanned for single TF-DNA binding affinities with TRAP and for each TF a ranked list of all promoters ordered by the binding affinity is created. We then study the similarity of the ranked lists and detect candidates for TF-TF interaction by applying a partial independence test for multiway contingency tables. Our candidates are validated by both known protein-protein interactions (PPIs) and known gene regulation mechanisms in the selected tissue. We find that the known PPIs are significantly enriched in the groups of our predicted TF-TF interactions (2 and 7 times more common than expected by chance). In addition, the predicted interacting TFs for studied tissues (liver, muscle, hematopoietic stem cell) are supported in literature to be active regulators or to be expressed in the corresponding tissue.
    [Show full text]
  • Genome-Wide Analysis of Saccharomyces Cerevisiae
    M BoC | ARTICLE Genome-wide analysis of Saccharomyces cerevisiae identifies cellular processes affecting intracellular aggregation of Alzheimer’s amyloid-β42: importance of lipid homeostasis S. Naira,*, M. Trainib,*, I. W. Dawesa,c, and G. G. Perroned aSchool of Biotechnology and Biomolecular Sciences and cRamaciotti Centre for Gene Function Analysis, University of New South Wales, Sydney, NSW 2052, Australia; bAtherosclerosis Laboratory, ANZAC Research Institute, Concord Hospital, Concord, NSW 2139, Australia; dSchool of Science and Health, University of Western Sydney, Penrith, NSW 1797, Australia ABSTRACT Amyloid-β (Aβ)–containing plaques are a major neuropathological feature of Al- Monitoring Editor zheimer’s disease (AD). The two major isoforms of Aβ peptide associated with AD are Aβ40 Charles Boone University of Toronto and Aβ42, of which the latter is highly prone to aggregation. Increased presence and aggre- gation of intracellular Aβ42 peptides is an early event in AD progression. Improved under- Received: Apr 26, 2013 standing of cellular processes affecting Aβ42 aggregation may have implications for develop- Revised: May 20, 2014 ment of therapeutic strategies. Aβ42 fused to green fluorescent protein (Aβ42-GFP) was Accepted: May 20, 2014 expressed in ∼4600 mutants of a Saccharomyces cerevisiae genome-wide deletion library to identify proteins and cellular processes affecting intracellular Aβ42 aggregation by assessing the fluorescence of Aβ42-GFP. This screening identified 110 mutants exhibiting intense Aβ42- GFP–associated fluorescence. Four major cellular processes were overrepresented in the data set, including phospholipid homeostasis. Disruption of phosphatidylcholine, phosphati- dylserine, and/or phosphatidylethanolamine metabolism had a major effect on intracellular Aβ42 aggregation and localization. Confocal microscopy indicated that Aβ42-GFP localiza- tion in the phospholipid mutants was juxtaposed to the nucleus, most likely associated with the endoplasmic reticulum (ER)/ER membrane.
    [Show full text]
  • Proteomic Identification of the Transcription Factors Ikaros And
    European School of Molecular Medicine (SEMM) University of Milan and University of Naples “Federico II” PhD degree in Systems Medicine (curriculum in Molecular Oncology) Settore disciplinare: BIO/11 Proteomic identification of the transcription factors Ikaros and Aiolos as new Myc interactors on chromatin Chiara Veronica Locarno Matricola: R10755 Center for Genomic Science IIT@SEMM, Milan Supervisor: Bruno Amati, PhD IEO, Milan Added Supervisor: Arianna Sabò, PhD IEO, Milan Academic year 2017-2018 Table of contents List of abbreviations ........................................................................................................... 4 List of figures ....................................................................................................................... 8 List of tables ....................................................................................................................... 11 Abstract .............................................................................................................................. 12 1. INTRODUCTION ......................................................................................................... 13 1.1 Myc ........................................................................................................................................ 13 1.1.1 Myc discovery and structure ........................................................................................... 13 1.1.2. Role of Myc in physiological and pathological conditions ...........................................
    [Show full text]
  • TASOR Is a Pseudo-PARP That Directs HUSH Complex Assembly and Epigenetic Transposon Control
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.974832; this version posted March 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control Christopher H. Douse1,‡, Iva A. Tchasovnikarova2,‡, Richard T. Timms2,‡, Anna V. Protasio2, Marta Seczynska2, Daniil M. Prigozhin1, Anna Albecka1,2, Jane Wagstaff3, James C. Williamson2, Stefan M.V. Freund3, Paul J. Lehner2*, Yorgo Modis1,2* 1 Molecular Immunity Unit, Department of Medicine, University of Cambridge, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK 2 Cambridge Institute of Therapeutic Immunology & Infectious Disease (CITIID), Department of Medicine, University of Cambridge, Cambridge CB2 0AW, UK 3 Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK Current addresses: Department of Experimental Medical Science, Lund University, Sölvegatan 19, Lund, Sweden (C.H.D.); The Gurdon Institute, Tennis Court Road, Cambridge, UK (I.A.T.); Department of Pathology, Tennis Court Road, Cambridge, UK (A.V.P.); Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA (D.M.P.) ‡These authors contributed equally *co-corresponding authors Keywords: Transcriptional repression; epigenetic silencing; antiretroviral response; genome stability; histone H3 lysine 9 methylation (H3K9me3); transposable element (TE); long interspersed nuclear element-1 (LINE-1); poly-ADP ribose polymerase (PARP); RNA-binding protein; RNA-induced transcriptional silencing; CUT&RUN; CUT&Tag; genome profiling 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.03.09.974832; this version posted March 11, 2020.
    [Show full text]
  • Transcriptional Events Co-Regulated by Hypoxia and Cold Stresses In
    Long et al. BMC Genomics (2015) 16:385 DOI 10.1186/s12864-015-1560-y RESEARCH ARTICLE Open Access Transcriptional events co-regulated by hypoxia and cold stresses in Zebrafish larvae Yong Long1, Junjun Yan1,2, Guili Song1, Xiaohui Li1,2, Xixi Li1,2, Qing Li1* and Zongbin Cui1* Abstract Background: Hypoxia and temperature stress are two major adverse environmental conditions often encountered by fishes. The interaction between hypoxia and temperature stresses has been well documented and oxygen is considered to be the limiting factor for the thermal tolerance of fish. Although both high and low temperature stresses can impair the cardiovascular function and the cross-resistance between hypoxia and heat stress has been found, it is not clear whether hypoxia acclimation can protect fish from cold injury. Results: Pre-acclimation of 96-hpf zebrafish larvae to mild hypoxia (5% O2) significantly improved their resistance to lethal hypoxia (2.5% O2) and increased the survival rate of zebrafish larvae after lethal cold (10°C) exposure. However, pre-acclimation of 96-hpf larvae to cold (18°C) decreased their tolerance to lethal hypoxia although their ability to endure lethal cold increased. RNA-seq analysis identified 132 up-regulated and 41 down-regulated genes upon mild hypoxia exposure. Gene ontology enrichment analyses revealed that genes up-regulated by hypoxia are primarily involved in oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. Hypoxia-inhibited genes are enriched in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process and cytidine deamination. A comparison with the dataset of cold-regulated gene expression identified 23 genes co-induced by hypoxia and cold and these genes are mainly associated with oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Functional Parsing of Driver Mutations in the Colorectal Cancer Genome Reveals Numerous Suppressors of Anchorage-Independent
    Supplementary information Functional parsing of driver mutations in the colorectal cancer genome reveals numerous suppressors of anchorage-independent growth Ugur Eskiocak1, Sang Bum Kim1, Peter Ly1, Andres I. Roig1, Sebastian Biglione1, Kakajan Komurov2, Crystal Cornelius1, Woodring E. Wright1, Michael A. White1, and Jerry W. Shay1. 1Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039. 2Department of Systems Biology, University of Texas M.D. Anderson Cancer Center, Houston, TX 77054. Supplementary Figure S1. K-rasV12 expressing cells are resistant to p53 induced apoptosis. Whole-cell extracts from immortalized K-rasV12 or p53 down regulated HCECs were immunoblotted with p53 and its down-stream effectors after 10 Gy gamma-radiation. ! Supplementary Figure S2. Quantitative validation of selected shRNAs for their ability to enhance soft-agar growth of immortalized shTP53 expressing HCECs. Each bar represents 8 data points (quadruplicates from two separate experiments). Arrows denote shRNAs that failed to enhance anchorage-independent growth in a statistically significant manner. Enhancement for all other shRNAs were significant (two tailed Studentʼs t-test, compared to none, mean ± s.e.m., P<0.05)." ! Supplementary Figure S3. Ability of shRNAs to knockdown expression was demonstrated by A, immunoblotting for K-ras or B-E, Quantitative RT-PCR for ERICH1, PTPRU, SLC22A15 and SLC44A4 48 hours after transfection into 293FT cells. Two out of 23 tested shRNAs did not provide any knockdown. " ! Supplementary Figure S4. shRNAs against A, PTEN and B, NF1 do not enhance soft agar growth in HCECs without oncogenic manipulations (Student!s t-test, compared to none, mean ± s.e.m., ns= non-significant).
    [Show full text]
  • PRODUCT SPECIFICATION Prest Antigen GTF2E2 Product Datasheet
    PrEST Antigen GTF2E2 Product Datasheet PrEST Antigen PRODUCT SPECIFICATION Product Name PrEST Antigen GTF2E2 Product Number APrEST89212 Gene Description general transcription factor IIE, polypeptide 2, beta 34kDa Alternative Gene FE, TF2E2, TFIIE-B Names Corresponding Anti-GTF2E2 (HPA004816) Antibodies Description Recombinant protein fragment of Human GTF2E2 Amino Acid Sequence Recombinant Protein Epitope Signature Tag (PrEST) antigen sequence: LLEDIEEALPNSQKAVKALGDQILFVNRPDKKKILFFNDKSCQFSVDEEF QKLWRSVTVDSMDEEKIEEYLKRQGISSMQESGPKKVAPIQRRKKPASQK KRRFKTHNEHLAGVLKDYSDITSSK Fusion Tag N-terminal His6ABP (ABP = Albumin Binding Protein derived from Streptococcal Protein G) Expression Host E. coli Purification IMAC purification Predicted MW 32 kDa including tags Usage Suitable as control in WB and preadsorption assays using indicated corresponding antibodies. Purity >80% by SDS-PAGE and Coomassie blue staining Buffer PBS and 1M Urea, pH 7.4. Unit Size 100 µl Concentration Lot dependent Storage Upon delivery store at -20°C. Avoid repeated freeze/thaw cycles. Notes Gently mix before use. Optimal concentrations and conditions for each application should be determined by the user. Product of Sweden. For research use only. Not intended for pharmaceutical development, diagnostic, therapeutic or any in vivo use. No products from Atlas Antibodies may be resold, modified for resale or used to manufacture commercial products without prior written approval from Atlas Antibodies AB. Warranty: The products supplied by Atlas Antibodies are warranted to meet stated product specifications and to conform to label descriptions when used and stored properly. Unless otherwise stated, this warranty is limited to one year from date of sales for products used, handled and stored according to Atlas Antibodies AB's instructions. Atlas Antibodies AB's sole liability is limited to replacement of the product or refund of the purchase price.
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]