Finetest Protein Recombinant Human HN1L
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Supporting Information
Supporting Information Edgar et al. 10.1073/pnas.1601895113 SI Methods (Actimetrics), and recordings were analyzed using LumiCycle Mice. Sample size was determined using the resource equation: Data Analysis software (Actimetrics). E (degrees of freedom in ANOVA) = (total number of exper- – Cell Cycle Analysis of Confluent Cell Monolayers. NIH 3T3, primary imental animals) (number of experimental groups), with −/− sample size adhering to the condition 10 < E < 20. For com- WT, and Bmal1 fibroblasts were sequentially transduced − − parison of MuHV-4 and HSV-1 infection in WT vs. Bmal1 / with lentiviral fluorescent ubiquitin-based cell cycle indicators mice at ZT7 (Fig. 2), the investigator did not know the genotype (FUCCI) mCherry::Cdt1 and amCyan::Geminin reporters (32). of the animals when conducting infections, bioluminescence Dual reporter-positive cells were selected by FACS (Influx Cell imaging, and quantification. For bioluminescence imaging, Sorter; BD Biosciences) and seeded onto 35-mm dishes for mice were injected intraperitoneally with endotoxin-free lucif- subsequent analysis. To confirm that expression of mCherry:: Cdt1 and amCyan::Geminin correspond to G1 (2n DNA con- erin (Promega E6552) using 2 mg total per mouse. Following < ≤ anesthesia with isofluorane, they were scanned with an IVIS tent) and S/G2 (2 n 4 DNA content) cell cycle phases, Lumina (Caliper Life Sciences), 15 min after luciferin admin- respectively, cells were stained with DNA dye DRAQ5 (abcam) and analyzed by flow cytometry (LSR-Fortessa; BD Biosci- istration. Signal intensity was quantified using Living Image ences). To examine dynamics of replicative activity under ex- software (Caliper Life Sciences), obtaining maximum radiance perimental confluent conditions, synchronized FUCCI reporter for designated regions of interest (photons per second per − − − monolayers were observed by time-lapse live cell imaging over square centimeter per Steradian: photons·s 1·cm 2·sr 1), relative 3 d (Nikon Eclipse Ti-E inverted epifluorescent microscope). -
Identification of Genomic Alterations in Castration Resistant Prostate Cancer Using Next Generation Sequencing
Identification of Genomic Alterations in Castration Resistant Prostate Cancer using Next Generation Sequencing Thesis Submitted for a Doctoral Degree in Natural Sciences (Dr. rer. nat) Faculty of Mathematics and Natural Sciences Rheinische Friedrich-Wilhelms- Submitted by Roopika Menon from Chandigarh, India Bonn 2013 Prepared with the consent of the Faculty of Mathematics and Natural Sciences at the Rheinische Friedrich-Wilhelms- 1. Reviewer: Prof. Dr. Sven Perner 2. Reviewer: Prof. Dr. Hubert Schorle Date of examination: 19 November 2013 Year of Publication: 2014 Declaration I solemnly declare that the work submitted here is the result of my own investigation, except where otherwise stated. This work has not been submitted to any other University or Institute towards the partial fulfillment of any degree. ____________________________________________________________________ Roopika Menon; Author Acknowledgements This thesis would not have been possible without the help and support of many people. I would like to dedicate this thesis to all the people who have helped make this dream a reality. This thesis would have not been possible without the patience, support and guidance of my supervisor, Prof. Dr. Sven Perner. It has truly been an honor to be his first PhD student. He has both consciously and unconsciously made me into the researcher that I am today. My PhD experience has truly been the ‘best’ because of his time, ideas, funding and most importantly his incredible sense of humor. He encouraged and gave me the opportunity to travel around the world to develop as a scientist. I cannot thank him enough for this immense opportunity, which stands as a stepping-stone to my career in science. -
Table S1. 103 Ferroptosis-Related Genes Retrieved from the Genecards
Table S1. 103 ferroptosis-related genes retrieved from the GeneCards. Gene Symbol Description Category GPX4 Glutathione Peroxidase 4 Protein Coding AIFM2 Apoptosis Inducing Factor Mitochondria Associated 2 Protein Coding TP53 Tumor Protein P53 Protein Coding ACSL4 Acyl-CoA Synthetase Long Chain Family Member 4 Protein Coding SLC7A11 Solute Carrier Family 7 Member 11 Protein Coding VDAC2 Voltage Dependent Anion Channel 2 Protein Coding VDAC3 Voltage Dependent Anion Channel 3 Protein Coding ATG5 Autophagy Related 5 Protein Coding ATG7 Autophagy Related 7 Protein Coding NCOA4 Nuclear Receptor Coactivator 4 Protein Coding HMOX1 Heme Oxygenase 1 Protein Coding SLC3A2 Solute Carrier Family 3 Member 2 Protein Coding ALOX15 Arachidonate 15-Lipoxygenase Protein Coding BECN1 Beclin 1 Protein Coding PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 Protein Coding SAT1 Spermidine/Spermine N1-Acetyltransferase 1 Protein Coding NF2 Neurofibromin 2 Protein Coding YAP1 Yes1 Associated Transcriptional Regulator Protein Coding FTH1 Ferritin Heavy Chain 1 Protein Coding TF Transferrin Protein Coding TFRC Transferrin Receptor Protein Coding FTL Ferritin Light Chain Protein Coding CYBB Cytochrome B-245 Beta Chain Protein Coding GSS Glutathione Synthetase Protein Coding CP Ceruloplasmin Protein Coding PRNP Prion Protein Protein Coding SLC11A2 Solute Carrier Family 11 Member 2 Protein Coding SLC40A1 Solute Carrier Family 40 Member 1 Protein Coding STEAP3 STEAP3 Metalloreductase Protein Coding ACSL1 Acyl-CoA Synthetase Long Chain Family Member 1 Protein -
Mouse Jpt2 Conditional Knockout Project (CRISPR/Cas9)
https://www.alphaknockout.com Mouse Jpt2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Jpt2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Jpt2 gene (NCBI Reference Sequence: NM_198937.2 ; Ensembl: ENSMUSG00000024165 ) is located on Mouse chromosome 17. 5 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 5 (Transcript: ENSMUST00000024981). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Jpt2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-40E8 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2 starts from about 7.89% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 4401 bp, and the size of intron 2 for 3'-loxP site insertion: 7237 bp. The size of effective cKO region: ~649 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 5 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Jpt2 Homology arm cKO region loxP site Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. -
Biomedical Informatics
BIOMEDICAL INFORMATICS Abstract GENE LIST AUTOMATICALLY DERIVED FOR YOU (GLAD4U): DERIVING AND PRIORITIZING GENE LISTS FROM PUBMED LITERATURE JEROME JOURQUIN Thesis under the direction of Professor Bing Zhang Answering questions such as ―Which genes are related to breast cancer?‖ usually requires retrieving relevant publications through the PubMed search engine, reading these publications, and manually creating gene lists. This process is both time-consuming and prone to errors. We report GLAD4U (Gene List Automatically Derived For You), a novel, free web-based gene retrieval and prioritization tool. The quality of gene lists created by GLAD4U for three Gene Ontology terms and three disease terms was assessed using ―gold standard‖ lists curated in public databases. We also compared the performance of GLAD4U with that of another gene prioritization software, EBIMed. GLAD4U has a high overall recall. Although precision is generally low, its prioritization methods successfully rank truly relevant genes at the top of generated lists to facilitate efficient browsing. GLAD4U is simple to use, and its interface can be found at: http://bioinfo.vanderbilt.edu/glad4u. Approved ___________________________________________ Date _____________ GENE LIST AUTOMATICALLY DERIVED FOR YOU (GLAD4U): DERIVING AND PRIORITIZING GENE LISTS FROM PUBMED LITERATURE By Jérôme Jourquin Thesis Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Biomedical Informatics May, 2010 Nashville, Tennessee Approved: Professor Bing Zhang Professor Hua Xu Professor Daniel R. Masys ACKNOWLEDGEMENTS I would like to express profound gratitude to my advisor, Dr. Bing Zhang, for his invaluable support, supervision and suggestions throughout this research work. -
Cutting the Brakes on Ras—Cytoplasmic Gaps As Targets of Inactivation in Cancer
cancers Review Cutting the Brakes on Ras—Cytoplasmic GAPs as Targets of Inactivation in Cancer Arianna Bellazzo and Licio Collavin * Department of Life Sciences, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy; [email protected] * Correspondence: [email protected] Received: 28 September 2020; Accepted: 15 October 2020; Published: 21 October 2020 Simple Summary: GTPase-Activating Proteins (RasGAPs) are a group of structurally related proteins with a fundamental role in controlling the activity of Ras in normal and cancer cells. In particular, loss of function of RasGAPs may contribute to aberrant Ras activation in cancer. Here we review the multiple molecular mechanisms and factors that are involved in downregulating RasGAPs expression and functions in cancer. Additionally, we discuss how extracellular stimuli from the tumor microenvironment can control RasGAPs expression and activity in cancer cells and stromal cells, indirectly affecting Ras activation, with implications for cancer development and progression. Abstract: The Ras pathway is frequently deregulated in cancer, actively contributing to tumor development and progression. Oncogenic activation of the Ras pathway is commonly due to point mutation of one of the three Ras genes, which occurs in almost one third of human cancers. In the absence of Ras mutation, the pathway is frequently activated by alternative means, including the loss of function of Ras inhibitors. Among Ras inhibitors, the GTPase-Activating Proteins (RasGAPs) are major players, given their ability to modulate multiple cancer-related pathways. In fact, most RasGAPs also have a multi-domain structure that allows them to act as scaffold or adaptor proteins, affecting additional oncogenic cascades. In cancer cells, various mechanisms can cause the loss of function of Ras inhibitors; here, we review the available evidence of RasGAP inactivation in cancer, with a specific focus on the mechanisms. -
WWOX Loses the Ability to Regulate Oncogenic AP-2Γ and Synergizes with Tumor Suppressor AP-2Α in High-Grade Bladder Cancer
cancers Article WWOX Loses the Ability to Regulate Oncogenic AP-2γ and Synergizes with Tumor Suppressor AP-2α in High-Grade Bladder Cancer Damian Kołat * , Zaneta˙ Kałuzi ´nska , Andrzej K. Bednarek and Elzbieta˙ Płuciennik Department of Molecular Carcinogenesis, Medical University of Lodz, 90-752 Lodz, Poland; [email protected] (Z.K.);˙ [email protected] (A.K.B.); [email protected] (E.P.) * Correspondence: [email protected] Simple Summary: A prognostic factor of bladder cancer which can benefit from research on molecu- lar markers is the histologic grade. WWOX, AP-2α and AP-2γ are known to have a grade-dependent effect but have not been investigated in that aspect in bladder cancer. Depending on the specific collaboration, their role was found to promote or inhibit grade 2 bladder cancer. As further research is needed on higher grades, the aim of the present study was to examine the functionality of WWOX and two AP-2 factors in grade 3 and grade 4 bladder cancer. It was found that WWOX, AP-2α and their combination mainly demonstrated anti-cancer properties; in contrast, AP-2γ promoted cancer development, which was not inhibited by WWOX in their combined variant. Next-generation sequencing was performed to identify the genes worth investigating as biomarkers. To conclude, WWOX and AP-2α demonstrate tumor suppressor synergism in high-grade bladder cancer, similar ˙ Citation: Kołat, D.; Kałuzi´nska, Z.; to intermediate grade. However, WWOX does not appear to guide oncogenic AP-2γ, which was the Bednarek, A.K.; Płuciennik, E. -
Unraveling the Cellular Origin and Clinical Prognostic Markers of Infant B-Cell Acute Lymphoblastic Leukemia Using Genome-Wide Analysis
Acute Lymphoblastic Leukemia SUPPLEMENTARY APPENDIX Unraveling the cellular origin and clinical prognostic markers of infant B-cell acute lymphoblastic leukemia using genome-wide analysis Antonio Agraz-Doblas, 1,2 Clara Bueno, 2# Rachael Bashford-Rogers, 3# Anindita Roy, 4,# Pauline Schneider, 5 Michela Bar - dini, 6 Paola Ballerini, 7 Gianni Cazzaniga, 6 Thaidy Moreno, 1 Carlos Revilla, 1 Marta Gut, 8,9 Maria G. Valsecchi, 10 Irene Roberts, 4,11 Rob Pieters, 5 Paola De Lorenzo, 10 Ignacio Varela, 1,$,* Pablo Menendez 2,12,13,$,* and Ronald W. Stam 5 1Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria-CSIC, Santander, Spain; 2Josep Carreras Leukemia Research Institute-Campus Clinic, Department of Biomedicine, School of Medicine, University of Barcelona, Spain; 3Department of Medicine, University of Cambridge, Cambridge Biomedical Campus, UK; 4Department of Paediatrics, University of Oxford, UK; 5Princess Maxima Center for Pediatric Oncology, Utrecht, the Netherlands; 6Centro Ricerca Tettamanti, Department of Pediatrics, University of Milano Bicocca, Fondazione MBBM, Monza, Italy; 7Pediatric Hematology, A. Trousseau Hospital, Paris, France; 8CNAG-CRG, Center for Genomic Regulation, Barcelona, Spain; 9Universitat Pompeu Fabra, Barcelona, Spain; 10 Interfant Trial Data Center, University of Milano-Bicocca, Monza, Italy; 11 MRC Molecular Haematology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, UK; 12 Instituciò Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain and 13 Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), ISCIII, Barcelona, Spain #These authors contributed equally to this work. $These senior authors contributed equally to this work. ©2019 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. -
Nuclear Receptor Co-Repressor Actions in Bladder Cancer
Nuclear receptor co-repressor actions in bladder cancer By Syed Asad Abedin A thesis presented to the School of Clinical and Experimental Medicine, University of Birmingham, For the degree of Doctor of Medicine (MD). School of Clinical and Experimental Medicine College of Medical and Dental Sciences University of Birmingham September 2009 1 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Summary Nuclear receptors are ligand dependent transcription factors and have defined expression patterns in classical tissues for example the Vitamin D receptor is expressed in small bowel, kidney and skin, as related to its function in maintaining serum calcium levels. However, nuclear receptor expression has also been demonstrated in non classical tissues for example VDR expression in the prostate and breast. Differential expression of a panel of nuclear receptors in bladder cancer cell lines (RT-4, RT-112, HT1376 and EJ-28 cells) at mRNA level and VDR and FXR expression at protein level has ben demonstrated. These cell lines demonstrate a range of anti-proliferative responses upon treatment with ligands to the panel of nuclear receptors (1α,25(OH)2D3, 9 cis RA, EPA, ETYA, CDA, LCA, 22HC, GW3965 and DHA). -
Analysis of Novel Steroidogenic Factor-1 Targets in the Human Adrenal Gland
Analysis of novel Steroidogenic Factor-1 targets in the human adrenal gland Bruno Ferraz de Souza UCL Institute of Child Health University College London Thesis submitted for the degree of Doctor of Philosophy (Ph.D.) 2011 Declaration I, Bruno Ferraz de Souza, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed ……………………………………………………. Date …………………. 2 Abstract Steroidogenic Factor-1 (SF-1, NR5A1 ) is a nuclear receptor transcription factor that plays a central role in adrenal and reproductive biology. In humans, SF-1 regulates adrenal development and disruption of SF-1 or its known targets is associated with impaired adrenal function. Therefore, the identification of novel SF-1 targets could reveal important new mechanisms in adrenal development and disease. This thesis describes three approaches to identifying SF-1 targets in NCI-H295R human adrenocortical cells. SF-1-dependent regulation of CITED2 and PBX1 was investigated since these factors regulate adrenal development in mice through pathways shared with Sf-1. Expression of CITED2 and PBX1 was confirmed in the developing human adrenal, and SF-1 was found to bind to and activate the CITED2 promoter and to cooperate with DAX1 to activate the PBX1 promoter. SF-1 binding was investigated using chromatin immunoprecipitation microarrays (ChIP-on-chip). These studies revealed that SF-1 binds to the extended promoter of 445 genes, including factors involved in angiogenesis. Angiopoietin 2 ( ANGPT2 ) emerged as a key novel SF-1 target, confirmed by transactivational studies, suggesting that regulation of angiogenesis might be an important additional action of SF-1 during adrenal development and tumorigenesis. -
Supplemental Material.Pdf
SUPPLEMENTAL MATERIAL FOR: Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia Grace J. Liu, Luisa Cimmino, Julian G. Jude, Yifang Hu, Matthew T. Witkowski, Mark D. McKenzie, Mutlu Kartal-Kaess, Sarah A. Best, Laura Tuohey, Yang Liao, Wei Shi, Charles G. Mullighan, Michael A. Farrar, Stephen L. Nutt, Gordon K. Smyth, Johannes Zuber, and Ross A. Dickins SUPPLEMENTAL FIGURE LEGENDS Figure S1. Restricted expression of Igh variable segments in STAT5-CA;Vav-tTA;TRE- GFP-shPax5 B-ALL. Expression (RNA-seq RPKM) of immunoglobulin heavy chain variable (Ighv) gene segments in leukemic cells sorted from untreated Rag1-/- mice (black) compared with normal pre-B cells (grey), showing dominant expression of Ighv2-5 in leukemia. Segments are arranged from 5’ to 3’. Mean ± SEM, n=3 mice for each group. Figure S2. Characterisation of the Pax5 restoration response of independent Stat5-CA; Vav-tTA; TRE-GFP-shPax5 leukemia A008. (A) Peripheral white blood cell (WBC) counts in Rag1–/– mice transplanted with leukemia cells from STAT5-CA;Vav-tTA;TRE-GFP-shPax5 mouse A008. Mean ± SEM, n=4 for each group prior to Dox treatment and following 14 days of Dox treatment as indicated. P < 0.0005, Student’s t test. (B) Flow cytometry of CD19 and IgM expression on mononuclear cells from the peripheral blood of a representative Rag1–/– mouse that was transplanted with leukemia A008 and subsequently Dox treated as indicated. (C) Flow cytometry of CD19 and IgD expression as shown in (B). (D) Leukemia burden (proportion of CD19+ cells in the blood) upon Dox treatment (mean ± SEM, n=4 mice). -
Methods for the Identification, Assessment
(19) TZZ__ _T (11) EP 1 581 629 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12Q 1/68 (2006.01) 01.04.2015 Bulletin 2015/14 (86) International application number: (21) Application number: 03796633.0 PCT/US2003/038539 (22) Date of filing: 04.12.2003 (87) International publication number: WO 2004/053066 (24.06.2004 Gazette 2004/26) (54) METHODS FOR THE IDENTIFICATION, ASSESSMENT, AND TREATMENT OF PATIENTS WITH PROTEASOME INHIBITION THERAPY VERFAHREN ZUR IDENTIFIZIERUNG, BEURTEILUNG UND BEHANDLUNG VON PATIENTEN MIT EINER PROTEASOMEN INHIBITIONS THERAPIE PROCEDESPOUR IDENTIFIER, EVALUER ET TRAITER DES PATIENTS SUIVANT UNE THERAPIE D’INHIBITION DE PROTEASOME (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • "Affymetrix GeneChip Human Genome U95 Set HU IE IT LI LU MC NL PT RO SE SI SK TR HG-U95A" GENBANK GEO, 11 March 2002 Designated Extension States: (2002-03-11), XP002293987 AL LT LV MK • AN W G ET AL: "PROTEASE INHIBITOR- INDUCED APOPTOSIS: ACCUMULATION OF WT (30) Priority: 06.12.2002 US 431514 P P53, P21WAF1/CIP1, AND INDUCTION OF APOPTOSIS ARE INDEPENDENT MARKERS OF (43) Date of publication of application: PROTEASOME INHIBITION" LEUKEMIA, 05.10.2005 Bulletin 2005/40 MACMILLAN PRESS LTD, US, vol. 14, no. 7, July 2000 (2000-07), pages 1276-1283, XP009081378 (73) Proprietor: MILLENNIUM PHARMACEUTICALS, ISSN: 0887-6924 INC. • AGHAJANIAN CAROL ET AL: "A phase I trial of Cambridge, MA 02139 (US) the novel proteasome inhibitor PS341 in advanced solid tumor malignancies." CLINICAL (72) Inventors: CANCER RESEARCH : AN OFFICIAL JOURNAL • MULLIGAN, George OFTHE AMERICAN ASSOCIATION FOR CANCER Lexington, MA 02421 (US) RESEARCH AUG 2002, vol.