HTRF Assays for Oncology and Inflammation

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HTRF assays for Oncology and Inflammation SIGNALING IN THE IMMUNE SYSTEM NK / NKT CELLS Cytokines Markers GM-CSF (CSF2) IL10 Granzyme B IFNg TNFa Perforin-1 Pi3 Syk AT ME SLP6 Iβ ER C-Raf NFkB Cisbio cell signaling assays available IMMUNE CHECKPOINTS Biochemical assays to identify checkpoint blockers TCR MHC II PD-1 PD-L1 CT C C CT C C G C MC II C SIα A2AR enosine T CELL SIGNALING TCR CD 4/8 CTLA4 PD1 CD3 CD28 CD40L AP0 SHP1 PLC SLP6 p38 c-Raf AT IP1 ME GS3β NFAT Iβ ER NFB AP1 nhiitory INγ I I I I TNα timulatory (Cisio cytokine assays availale) DNA Cisio cell signaling assays availale pSLP6 Dexamethasone inhibitory effect on CD3/CD28 stimulated PBMCs 8000 10000 ** hIFNγ L m 6000 / g hIL2 p ] s 5000 4000 e n 2000 i k o t HTRF signal 2000 y C 1000 Neg [ 0 0 Basal 4 40 CD3/CD28 anti-CD3 Ab (g/mL) Dexamethasone Pathway monitoring in primary T cells upon CD3 stimulation Cytokine panel on PBMCs (300,000 cells per well, 5 min CD3 stimulation) B CELL SIGNALING BCR PD1 CD28 CTLA4 CD40L BT SHP1 AT PLC BLN Iβ GS3β c-Raf IP1 ME NFAT NFB ER nhiitory AP1 INγ I I I I I TNα timulatory (Cisio cytokine assays availale) DNA Cisio cell signaling assays availale Stimulation effect of pervanadate on Raji cells Dexamethasone inhibitory effect on LPS stimlated PBMCs 15000 S/B = 10.7 40000 25000 o H h 20000 T hIL6 L p R s 30000 m / F o 15000 10000 g hIL1β h R p P a ] t 10000 s o i i 20000 hTNFα o t e 4000 a n T i o R k 5000 t o F a 10000 t y R l 2000 T C [ H 0 0 0 0 15 30 60 0 15 30 60 LPS [Pervanadate] (µM) Dexamethasone Pervanadate effect on BTK accumulation (phospho & total) Cytokine panel on PBMCs INNATE IMMUNITY Cytokines Inflammation markers IL1b CCL3 (MIP1a) Histamine IL6 CCL4 (MIP1b) PGE2 IL8 CCL5 (RANTES) LTC4 IFNb TNFa LTB4 CXCL10 (IP-10) TLR5 TLR10 TLR2/TLR6 TLR1/TLR2 TLR4 dsDNA cGAS MYD88 ATP + GTP 2’-3’ cGAMP TBK1 IRAK3 IRAK1 IRAK2 STING IRAK4 IRF3 STING p38 IKKβ TAK1 JNK NFκB NFκB IRF3 IRF3 TNFα, IL6, IL1β Type-I-IFNs Cisio cell signaling assays availale Toll-like receptor STING signaling pathway signaling pathway CELLULAR PHOSPHORYLATION ASSAYS Transfer ncuate supernatant Plate timulate T detection 3 yse antiodies • Cell-based assays • Compatible with cell lysates, tissue extracts, iPS cells, 3D cell models • Sample volume 16 μL • More sensitive than Western Blot • < 1 hr bench time • Pharmacological studies of pathway inhibitors, agonists, and allosteric modulators CELL-BASED CYTOKINE ASSAYS ncuate sample T detection antiodies • Sample volume 16 μL • < 2 hrs bench time • Validated on PBMCs and cell lines (THP1, Jurkat...) • Compatible with Cisbio 96-well low-volume plate • Allow multiple cytokine measurements from 1 sample MDB008 - 01/2020 - Copyright Cisbio Bioassays. All rights reserved. HTRF and Tag-lite are registered trademarks of Cisbio Bioassays. Europe and other countries +33(0)466 796 705 | [email protected] U.S.A. and Canada 1-888-963-4567 | [email protected] China +86 21 5018 9880 | [email protected] Japan +81-(0)43-306-8712 | [email protected] www.cisbio.com.

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Carna Newsletter 2021.05.26
画像：ファイル＞配置＞リンクから画像を選択＞画像を選択して右クリック＞画像を最前面に配置＞オブジェクト＞テキストの回り込み＞作成 Vol.10 Carna Newsletter 2021.05.26 Targeted Degradation of Non-catalytic Kinases New Drug Discovery Options The announcement that Kymera Therapeutics, a TLR/IL-1R signaling in several cell types, which company pioneering targeted protein degradation, indicates that the IRAK4 scaffolding function is entered into a strategic collaboration with Sanofi important in some cases2)3). to develop and commercialize first-in-class protein degrader therapies targeting IRAK4 in patients with immune-inflammatory diseases highlights the TLR growing interest in clinical applications of small IL-1R molecule mediated kinase degradation. Kymera received $150 million in cash up front and may cytosol 8 8 potentially receive at least $2 billion in this key D y strategic partnership, including sales milestones M 4 4 K K and royalty payments. A A R R I I 2 P 1 K Most of the protein degraders currently under K A A R I R development are heterobifunctional molecules I which contain one moiety that binds a desired target protein and another that binds an E3 ligase, P joined by a linker. Protein degrader-induced proximity results in ubiquitination of the target Fig.1. TLR and IL-1R Signaling in the Myddosome followed by its degradation by the proteasome. This transformative new modality is expected to open a new chapter in drug discovery targeting 【Allosteric regulatory function】 kinases for which the development of clinical Aside from scaffolding functions, allosteric inhibitors has been difficult. One such example is regulation is another non-catalytic function that IRAK4, which has a non-catalytic function activates binding partners by inducing a independent of kinase activity in addition to a conformational change.
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Supplementary Material
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry Page 1 / 45 SUPPLEMENTARY MATERIAL Appendix A1: Neuropsychological protocol. Appendix A2: Description of the four cases at the transitional stage. Table A1: Clinical status and center proportion in each batch. Table A2: Complete output from EdgeR. Table A3: List of the putative target genes. Table A4: Complete output from DIANA-miRPath v.3. Table A5: Comparison of studies investigating miRNAs from brain samples. Figure A1: Stratified nested cross-validation. Figure A2: Expression heatmap of miRNA signature. Figure A3: Bootstrapped ROC AUC scores. Figure A4: ROC AUC scores with 100 different fold splits. Figure A5: Presymptomatic subjects probability scores. Figure A6: Heatmap of the level of enrichment in KEGG pathways. Kmetzsch V, et al. J Neurol Neurosurg Psychiatry 2021; 92:485–493. doi: 10.1136/jnnp-2020-324647 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry Appendix A1. Neuropsychological protocol The PREV-DEMALS cognitive evaluation included standardized neuropsychological tests to investigate all cognitive domains, and in particular frontal lobe functions. The scores were provided previously (Bertrand et al., 2018). Briefly, global cognitive efficiency was evaluated by means of Mini-Mental State Examination (MMSE) and Mattis Dementia Rating Scale (MDRS). Frontal executive functions were assessed with Frontal Assessment Battery (FAB), forward and backward digit spans, Trail Making Test part A and B (TMT-A and TMT-B), Wisconsin Card Sorting Test (WCST), and Symbol-Digit Modalities test.
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Supplementary Material DNA Methylation in Inflammatory Pathways Modifies the Association Between BMI and Adult-Onset Non- Atopic
Supplementary Material DNA Methylation in Inflammatory Pathways Modifies the Association between BMI and Adult-Onset Non- Atopic Asthma Ayoung Jeong 1,2, Medea Imboden 1,2, Akram Ghantous 3, Alexei Novoloaca 3, Anne-Elie Carsin 4,5,6, Manolis Kogevinas 4,5,6, Christian Schindler 1,2, Gianfranco Lovison 7, Zdenko Herceg 3, Cyrille Cuenin 3, Roel Vermeulen 8, Deborah Jarvis 9, André F. S. Amaral 9, Florian Kronenberg 10, Paolo Vineis 11,12 and Nicole Probst-Hensch 1,2,* 1 Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland; [email protected] (A.J.); [email protected] (M.I.); [email protected] (C.S.) 2 Department of Public Health, University of Basel, 4001 Basel, Switzerland 3 International Agency for Research on Cancer, 69372 Lyon, France; [email protected] (A.G.); [email protected] (A.N.); [email protected] (Z.H.); [email protected] (C.C.) 4 ISGlobal, Barcelona Institute for Global Health, 08003 Barcelona, Spain; [email protected] (A.-E.C.); [email protected] (M.K.) 5 Universitat Pompeu Fabra (UPF), 08002 Barcelona, Spain 6 CIBER Epidemiología y Salud Pública (CIBERESP), 08005 Barcelona, Spain 7 Department of Economics, Business and Statistics, University of Palermo, 90128 Palermo, Italy; [email protected] 8 Environmental Epidemiology Division, Utrecht University, Institute for Risk Assessment Sciences, 3584CM Utrecht, Netherlands; [email protected] 9 Population Health and Occupational Disease, National Heart and Lung Institute, Imperial College, SW3 6LR London, UK; [email protected] (D.J.); [email protected] (A.F.S.A.) 10 Division of Genetic Epidemiology, Medical University of Innsbruck, 6020 Innsbruck, Austria; [email protected] 11 MRC-PHE Centre for Environment and Health, School of Public Health, Imperial College London, W2 1PG London, UK; [email protected] 12 Italian Institute for Genomic Medicine (IIGM), 10126 Turin, Italy * Correspondence: [email protected]; Tel.: +41-61-284-8378 Int.
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And IRAK1 Knock-In Mice Production Defined by the Study of IRAK2 Two
Two Phases of Inflammatory Mediator Production Defined by the Study of IRAK2 and IRAK1 Knock-in Mice This information is current as Eduardo Pauls, Sambit K. Nanda, Hilary Smith, Rachel of September 29, 2021. Toth, J. Simon C. Arthur and Philip Cohen J Immunol 2013; 191:2717-2730; Prepublished online 5 August 2013; doi: 10.4049/jimmunol.1203268 http://www.jimmunol.org/content/191/5/2717 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/08/06/jimmunol.120326 Material 8.DC1 http://www.jimmunol.org/ References This article cites 55 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/191/5/2717.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/191/10/5317.full.pdf The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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IRAK2, Active IRAK2, Active
Catalogue # Aliquot Size I10-10BG-05 5 µg I10-10BG-10 10 µg I10-10BG-20 20 µg IRAK2, Active Full-length recombinant protein expressed in Sf9 cells Catalog # I10-10BG Lot # V117 -2B Product Description Specific Activity Recombinant full-length human IRAK2 was expressed by 280,000 baculovirus in Sf9 insect cells using an N-terminal GST tag. The gene accession number is NM_001570 . 210,000 Gene Aliases 140,000 IRAK-2, MGC150550 70,000 (RLU) Activity Formulation 0 0 200 400 600 800 Recombinant protein stored in 50mM Tris-HCl, pH 7.5, Protein (ng) 150mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25mM DTT, 0.1mM PMSF, 25% glycerol. The specific activity of IRAK2 was determined to be 6 nmol /min/mg as per activity assay protocol. Storage and Stability Purity o Store product at –70 C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. The purity of IRAK2 was determined to be >75% by densitometry, approx. MW 103kDa . Scientific Background Interleukin-1 receptor-associated kinase 2 (IRAK2) is important downstream signaling components of Toll-like receptors (TLRs) (1). IRAKs were first described as signal transducers for IL-1 and later have been implicated in signal transduction of other members of the Toll/IL-1 IRAK2, Active receptor family. The interleukin-1 receptor (IL-1R) signaling Full-length recombinant protein expressed in Sf9 cells pathway leads to NF κB activation in mammals. To date, Catalog Number I10-10BG four mammalian IRAKs have been identified (IRAK-1, Specific Activity 6 nmol/min/mg IRAK-2, IRAK-4, and IRAK-M) (2).
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IRAK2 Directs Stimulus-Dependent Nuclear Export of Inflammatory Mrnas
IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Zhou, H., K. Bulek, X. Li, T. Herjan, M. Yu, W. Qian, H. Wang, et al. 2017. “IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.” eLife 6 (1): e29630. doi:10.7554/eLife.29630. http://dx.doi.org/10.7554/eLife.29630. Published Version doi:10.7554/eLife.29630 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:34493075 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA RESEARCH ARTICLE IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs Hao Zhou1†, Katarzyna Bulek1,2†, Xiao Li3†, Tomasz Herjan1†, Minjia Yu1,4, Wen Qian1, Han Wang1, Gao Zhou3, Xing Chen1, Hui Yang1, Lingzi Hong1, Junjie Zhao1, Luke Qin1, Koichi Fukuda5, Annette Flotho6, Ji Gao7, Ashok Dongre7, Julie A Carman7, Zizhen Kang1,8,9, Bing Su8,9,10, Timothy S Kern11,12, Jonathan D Smith13, Thomas A Hamilton1, Frauke Melchior6, Paul L Fox13, Xiaoxia Li1* 1Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, United States; 2Department of Immunology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 3Department of Genetics, Stanford University School of Medicine,
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And Multiple Conformations Kinase 4 Structures Reveal Novel Features Cutting Edge: IL-1 Receptor-Associated
Cutting Edge: IL-1 Receptor-Associated Kinase 4 Structures Reveal Novel Features and Multiple Conformations This information is current as Andreas Kuglstatter, Armando G. Villaseñor, David Shaw, of September 23, 2021. Simon W. Lee, Stan Tsing, Linghao Niu, Kyung W. Song, Jim W. Barnett and Michelle F. Browner J Immunol 2007; 178:2641-2645; ; doi: 10.4049/jimmunol.178.5.2641 http://www.jimmunol.org/content/178/5/2641 Downloaded from References This article cites 23 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/178/5/2641.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 23, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. THE JOURNAL OF IMMUNOLOGY CUTTING EDGE Cutting Edge: IL-1 Receptor-Associated Kinase 4 Structures Reveal Novel Features and Multiple Conformations Andreas Kuglstatter,1 Armando G. Villasen˜or, David Shaw, Simon W.
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Crystal Structure of Human IRAK1
Crystal structure of human IRAK1 Li Wanga,b, Qi Qiaoa,b, Ryan Ferraoa,b, Chen Shena,b, John M. Hatchera,c, Sara J. Buhrlagea,c, Nathanael S. Graya,c, and Hao Wua,b,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; and cDepartment of Cancer Biology, Dana–Farber Cancer Institute, Boston, MA 02115 Edited by Jonathan C. Kagan, Boston Children’s Hospital, Boston, MA, and accepted by Editorial Board Member K. C. Garcia November 7, 2017 (received for review August 14, 2017) Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/ activity, enabling subsequent IRAK1 autophosphorylation in its ac- threonine kinases that play critical roles in initiating innate immune tivation loop to become fully activated (11). responses against foreign pathogens and other types of dangers Active IRAK1 undergoes further autophosphorylation, lead- through their role in Toll-like receptor (TLR) and interleukin 1 receptor ing to hyperphosphorylation of the ProST region. This hyper- (IL-1R) mediated signaling pathways. Upon ligand binding, TLRs and phosphorylation induces IRAK1 to dissociate from the Myddosome IL-1Rs recruit adaptor proteins, such as myeloid differentiation and associate with the downstream effector TRAF6 (1, 12). TRAF6 primary response gene 88 (MyD88), to the membrane, which in turn activation and ubiquitination of IRAK1 trigger the initiation of a recruit IRAKs via the death domains in these proteins to form the transcriptional program mediated by NF-κB and activator protein 1, Myddosome complex, leading to IRAK kinase activation. Despite leading to the induction of proinflammatory and immunomodula- their biological and clinical significance, only the IRAK4 kinase tory cytokines, such as IL-1β,TNF-α, IL-6, and IL-18.
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Point Mutation Specific Antibodies in B-Cell and T-Cell Lymphomas And
diagnostics Review Point Mutation Specific Antibodies in B-Cell and T-Cell Lymphomas and Leukemias: Targeting IDH2, KRAS, BRAF and Other Biomarkers RHOA, IRF8, MYD88, ID3, NRAS, SF3B1 and EZH2 Kunwar Singh 1,*,† , Sumanth Gollapudi 2,† , Sasha Mittal 2, Corinn Small 2, Jyoti Kumar 1 and Robert S. Ohgami 2,* 1 Department of Pathology, Stanford University, Stanford, CA 94063, USA; [email protected] 2 Department of Pathology, University of California, San Francisco, CA 94143, USA; [email protected] (S.G.); [email protected] (S.M.); [email protected] (C.S.) * Correspondence: [email protected] (K.S.); [email protected] (R.S.O.); Tel.: +1-347-856-7047 (K.S.); +1-415-514-8179 (R.S.O.) † These authors contributed equally. Abstract: B-cell and T-cell lymphomas and leukemias often have distinct genetic mutations that are diagnostically defining or prognostically significant. A subset of these mutations consists of specific point mutations, which can be evaluated using genetic sequencing approaches or point mutation Citation: Singh, K.; Gollapudi, S.; specific antibodies. Here, we describe genes harboring point mutations relevant to B-cell and T-cell Mittal, S.; Small, C.; Kumar, J.; malignancies and discuss the current availability of these targeted point mutation specific antibodies. Ohgami, R.S. Point Mutation Specific We also evaluate the possibility of generating novel antibodies against known point mutations Antibodies in B-Cell and T-Cell by computationally assessing for chemical and structural features as well as epitope antigenicity Lymphomas and Leukemias: of these targets. Our results not only summarize several genetic mutations and identify existing Targeting IDH2, KRAS, BRAF and point mutation specific antibodies relevant to hematologic malignancies, but also reveal potential Other Biomarkers RHOA, IRF8, underdeveloped targets which merit further study.
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Crystal Structure of Human IRAK1
Crystal structure of human IRAK1 Li Wanga,b, Qi Qiaoa,b, Ryan Ferraoa,b, Chen Shena,b, John M. Hatchera,c, Sara J. Buhrlagea,c, Nathanael S. Graya,c, and Hao Wua,b,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bProgram in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA 02115; and cDepartment of Cancer Biology, Dana–Farber Cancer Institute, Boston, MA 02115 Edited by Jonathan C. Kagan, Boston Children’s Hospital, Boston, MA, and accepted by Editorial Board Member K. C. Garcia November 7, 2017 (received for review August 14, 2017) Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/ activity, enabling subsequent IRAK1 autophosphorylation in its ac- threonine kinases that play critical roles in initiating innate immune tivation loop to become fully activated (11). responses against foreign pathogens and other types of dangers Active IRAK1 undergoes further autophosphorylation, lead- through their role in Toll-like receptor (TLR) and interleukin 1 receptor ing to hyperphosphorylation of the ProST region. This hyper- (IL-1R) mediated signaling pathways. Upon ligand binding, TLRs and phosphorylation induces IRAK1 to dissociate from the Myddosome IL-1Rs recruit adaptor proteins, such as myeloid differentiation and associate with the downstream effector TRAF6 (1, 12). TRAF6 primary response gene 88 (MyD88), to the membrane, which in turn activation and ubiquitination of IRAK1 trigger the initiation of a recruit IRAKs via the death domains in these proteins to form the transcriptional program mediated by NF-κB and activator protein 1, Myddosome complex, leading to IRAK kinase activation. Despite leading to the induction of proinflammatory and immunomodula- their biological and clinical significance, only the IRAK4 kinase tory cytokines, such as IL-1β,TNF-α, IL-6, and IL-18.
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Targeting CDK4 Overcomes EMT-Mediated Tumor Heterogeneity and Therapeutic Resistance in KRAS Mutant Lung Cancer
Targeting CDK4 overcomes EMT-mediated tumor heterogeneity and therapeutic resistance in KRAS mutant lung cancer Aparna Padhye1,2, Jessica M. Konen1, B. Leticia Rodriguez1, Jared J. Fradette1, Joshua K. Ochieng1, Lixia Diao3, Jing Wang3, Wei Lu4, Luisa S. Solis4, Harsh Batra4, Maria G. Raso4, Michael D. Peoples5, Rosalba Minelli5, Alessandro Carugo5, Christopher A. Bristow5, Don L. Gibbons1,6* 1. Department of Thoracic/Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 2. University of Texas Graduate School of Biomedical Sciences, Houston, TX 77030, USA. 3. Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 4. Department of Translational Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 5. TRACTION Platform, Division of Therapeutics Development, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 6. Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. *Corresponding author. Email: [email protected] Supplemental Methods Plasmids, Transfections, and Lentiviral Generation and Transduction Transfections of si-RNAs werr performed using the Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). Constitutive Cdkn1a overexpression cell lines were generated by using Cdkn1a mouse Tagged ORF Clone (Origene (NM_007669)). Cdkn1a ORF was also subcloned into dox-inducible pTRIPZ-GFP vector to generate doxycycline inducible cell lines using EcoRI and AgeI restriction cut sites. Constitutive Cdkn1a shRNAs were purchased from Milipore sigma. The sequences used in the experiments are listed in table S11. Dox- inducible shRNAs were expressed in Tet-pLKO-puro vector with a scramble sequence as the non-targeting control.
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Kinome Expression Profiling to Target New Therapeutic Avenues in Multiple Myeloma
Plasma Cell DIsorders SUPPLEMENTARY APPENDIX Kinome expression profiling to target new therapeutic avenues in multiple myeloma Hugues de Boussac, 1 Angélique Bruyer, 1 Michel Jourdan, 1 Anke Maes, 2 Nicolas Robert, 3 Claire Gourzones, 1 Laure Vincent, 4 Anja Seckinger, 5,6 Guillaume Cartron, 4,7,8 Dirk Hose, 5,6 Elke De Bruyne, 2 Alboukadel Kassambara, 1 Philippe Pasero 1 and Jérôme Moreaux 1,3,8 1IGH, CNRS, Université de Montpellier, Montpellier, France; 2Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussels, Belgium; 3CHU Montpellier, Laboratory for Monitoring Innovative Therapies, Department of Biologi - cal Hematology, Montpellier, France; 4CHU Montpellier, Department of Clinical Hematology, Montpellier, France; 5Medizinische Klinik und Poliklinik V, Universitätsklinikum Heidelberg, Heidelberg, Germany; 6Nationales Centrum für Tumorerkrankungen, Heidelberg , Ger - many; 7Université de Montpellier, UMR CNRS 5235, Montpellier, France and 8 Université de Montpellier, UFR de Médecine, Montpel - lier, France ©2020 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2018.208306 Received: October 5, 2018. Accepted: July 5, 2019. Pre-published: July 9, 2019. Correspondence: JEROME MOREAUX - [email protected] Supplementary experiment procedures Kinome Index A list of 661 genes of kinases or kinases related have been extracted from literature9, and challenged in the HM cohort for OS prognostic values The prognostic value of each of the genes was computed using maximally selected rank test from R package MaxStat. After Benjamini Hochberg multiple testing correction a list of 104 significant prognostic genes has been extracted. This second list has then been challenged for similar prognosis value in the UAMS-TT2 validation cohort.
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