DOCSLIB.ORG

Supporting Information Supporting Information Corrected July 15 , 2014 Yi Et Al

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Supporting Information Supporting Information Corrected July 15 , 2014 Yi Et Al Supporting Information Supporting Information Corrected July 15 , 2014 Yi et al. 10.1073/pnas.1404943111 SI Materials and Methods which no charge state could be determined were excluded MS/ Tumorsphere Growth Assay. Single-cell tumorsphere growth assays MS selection). Three independent experiments were performed. of human mammary epithelial (HMLER) (CD44high/CD24low)SA and HMLER (CD44high/CD24low)FA population cells were per- Phosphorylation Site Localization. The probability of correct phos- formed with ultra-low attachment surface six-well plates (Corn- phorylation site localization for each phosphorylation site was ing) and the mammary epithelial cell basal medium (MEBM) measured using an Ascore algorithm (1). This algorithm con- medium (Lonza) was supplemented with B27 (Invitrogen), 20 ng/mL siders all phosphoforms of a peptide and uses the presence or EGF, and 20 ng/mL basic FGF (BD Biosciences), and 4 μg/mL absence of experimental fragment ions unique to each to create an ambiguity score (Ascore). Parameters included a window size of heparin (Sigma). Single cells were cultured for 8 d and images were ± taken with a Nikon camera. 100 m/z units and a fragment ion tolerance of 0.6 m/z units. Sites with an Ascore of ≥13 (P ≤ 0.05) were considered to be ≥ ≤ Soft Agar Colony Growth Assay. Single-cell soft agar colony for- confidently localized, and those with an Ascore of 19 (P 0.01) mation and growth assays were performed to identify the tumor- were considered to be localized with near certainty. More than ≥ igenesis capacity of HMLER (CD44high/CD24low)SA and HMLER a 2.5-fold ratio ( 60.02%) was considered as a significant (CD44high/CD24low)FA population cells in vitro. Briefly, base agar phosphorylation increase in phosphoproteome assays. As cellu- layer was 0.5% agar with MEBM, the top agar layer was 0.35% agar lar active kinases are rapidly dephosphorylated by phospha- ∼ with mammary epithelial cell growth medium (MEGM) (500 mL tases in cells (2, 3), 24 kinases within 1.6- to 2-fold ratio (an ∼ MEBMwith2mLBPE,0.5mLinsulin,0.5mLrhEGF,0.5mL increase from 37.5% to 50%) were tracked and recovered as GA-1000, 0.5 mL hydrocortisone; Lonza). HMLER (CD44high/ phosphorylation increased kinases. A decrease of more than CD24low)SA and HMLER (CD44high/CD24low)FA population cells 60% was considered a significant phosphorylation decrease. (1 × 104) in single-cell suspensions were mixed with 2 mL 0.35% The postsearch data analyses were performed as previously de- agar with MEGM, and then the mixture was added onto the base scribed (4). agar layer of each well in the six-well plates. After about 2 wk, Database Searches, Data Filtering, Validation of Protein Detection grossly visible colonies appeared. Images were taken with a Nikon Rate, and Phosphoproteome Analyses. The spectral data were camera. Colony diameters were analyzed and numbers counted searched with SEQUEST (1) against a database containing the with ImageJ software (http://imagej.nih.gov/ij/). human protein sequence database (www.ensembl.org/index.html) high low SA together with the reversed complement. The LC-MS/MS identi- Cell Viability Assay. HMLER (CD44 /CD24 ) and HMLER (CD44high/CD24low)FA population cells (1 × 104) were treated fications were filtered to 0.98% protein false discovery rate (FDR) and 0.1% peptide FDR. The peptide quantification and with indicated compounds with series of concentrations for 24 h. phosphorylation site localization were analyzed using in-house Cell viability assays were performed on the cells with CellTiter- software and Ascore as previously described (5). Glo luminescent cell viability assay kit (Promega) according to the manual. Three independent experiments were performed. Peptide Quantification. Peptide quantification was performed us- ing the Vista program. We required a signal-to-noise ratio (S/N) Strong Cation Exchange/Immobilized Metal Affinity Chromatography value >3 for both heavy and light species for quantification. For Phosphopeptide Enrichment and Liquid Chromatography Tandem peptides found exclusively as singlets (only a heavy or only a light Mass Spectrometry Analyses. In phosphopeptide enrichment by peak present), we reported the peak S/N ratio or its inverse, as strong cation exchange/immobilized metal affinity chromatog- a proxy for relative abundance measurement. For such peptides, raphy (SCX/IMAC), peptides were first separated at acidic pH on we required an S/N value >5 for the observed species. In addi- a polysulfethyl A semipreparative column (9.4 mm i.d. × 200 mm tion, if the S/N value of one member of a pair was <3, the length, 5-μm particle size, 200-Å pore size; PolyLC). Twelve partner value was required to be >5. Finally, to avoid quantifying fractions were collected and further enriched by IMAC, starting false positives, any identification from a singlet peak was re- with 30 μL of PHOS-Select beads (Sigma-Aldrich). Enriched quired to pass an identification threshold that was 10 times more phosphopeptides were eluted and desalted by STAGE-TIPS stringent (Q value of <0.001; precision >99.9%). Raw abun- (Thermo Scientific) as previously described (1). Phosphopep- dance ratios from each experiment were normalized based on tide-enriched fractions were analyzed by liquid chromatography the median distribution ratio. tandem mass spectrometry (LC-MS/MS) on an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) equipped with Accessibility of Raw Data and Bioinformatic Analysis. The five Excel a Thermo Fisher Scientific nanospray source, an Agilent 1100 files (Files S1–S5) and MS raw data are available at http:// Series binary HPLC pump, and a Famos autosampler. Phos- gwagner.med.harvard.edu/intranet/PNAS_Manuscript_2014/. phopeptides were separated on a 0.125 × 180 mm fused silica microcapillary column with an in needle tip (made in-house) Western Blot Assay. Total proteins were extracted from breast with a ∼5-μm i.d. The silica microcapillary column was packed cancer stem cells (CSCs) with protease inhibitor (Roche) and with magicC18AQ C18 reverse-phase resin (5-μm particle size, phosphatase inhibitor (Sigma) for Western blot assays with 200-Å pore size; Michrom Bioresources). Separation was per- specific antibodies. Anti-p120 catenin and anti–p-p120 (Tyr280) formed by applying a 57-min gradient from 7% to 28% aceto- catenin antibodies were obtained from Syd Labs. Anti-GSK3β, nitrile in 0.125% formic acid. The mass spectrometer was anti-p(Ser9) GSK3β, anti-PKAC, anti-p(Thr197) PKAC, anti- operated with default settings: full MS [automatic gain control ERK1/2, anti-p(Thr202/Tyr204)-ERK1/2, anti-p(Ser217/221) (AGC), 1 × 106; resolution, 6 × 104; m/z range, 375–1,800; MEK1/2, anti-MEK1/2, anti-chemokine (C-X-C motif) re- maximum ion time, 1,000 ms]; MS/MS (AGC, 5 × 103; maximum ceptor 4 (CXCR4), anti–p-Ser/Thr, anti–β-actin, and anti-tubulin ion time, 120 ms; minimum signal threshold, 4 × 103; dynamic antibodies were ordered from Cell Signaling. Anti–p-Tyr (mono- exclusion time setting, 30 s; singly charged ions and ions for clonal) antibodies were ordered from Abcam. PKA inhibitor Yi et al. www.pnas.org/cgi/content/short/1404943111 1of15 (14-22-Amide) was ordered from EMD. AMD3100 (A5602) was (http://networkin.info/search.php), and Kinasource (www.kinasource. ordered from Sigma. The short hairpin RNA (shRNA)-CXCR4 co.uk/Database/substrates.html) (9), phosphotase–substrates with plasmids and control shRNA plasmids were ordered from The PubMed, and pathways with the Kyoto Encyclopedia of Genes RNAi Consortium (Broad Institute). Active SDF-1 protein was and Genomes (www.genome.jp/kegg/pathway.html). The kinase– obtained from Abcam (ab78808). Each experiment was repeated phosphorylation site-specific substrate relationships were either at least three times. supplied in Kinasource or reported in the literature (Table S2, Cellular protein extraction and Western blot analysis was S3, and S7). All relationships of the kinase–substrate and performed with radioimmunoprecipitation assay buffer (50 mM phosphotase–substrate in this study are experimentally identified Tris·HCl pH 7.4, 150 mM NaCl, 1% Triton-X100, 0.1% SDS, × in human cells in previously published reports (refs. 5, 9, 10, and 0.25% Na-deoxycholate, 1 mM PMSF, 1 Roche complete mini 11 and Tables S3 and S7). The results were compared with 40 protease inhibitor mixture, 1× Pierce phosphatase inhibitor phosphoproteomic experiments of mammalian cells in the lit- mixture). The total Ser/Thr, or Tyr phosphorylation signal in- erature to validate the prediction for the biological function, tensities in Western blots were analyzed by Bio-Rad imaging system analysis software. The ratios of phosphorylation signal activation, and/or inhibition of phosphorylation sites. The net- intensities of samples compared with the control were expressed works were seeded on established SDF-1/CXCR4 signaling – as relative phosphorylation activities. pathways (12 14) with extended signal transduction based on the above-mentioned integrated bioinformatic analyses of phospho- Bioinformatic Analysis. Bioinformatic annotations were carried out proteins in the phosphoproteome. using Human Protein Reference Database (www.hprd.org)as previously described (4, 6). The phosphosites were identified Conserved Phosphorylation Site Analysis Across Species. Conserved using the Web-based program www.phosphosite.org and phosphorylation sites and motifs were analyzed with target PubMed database (www.ncbi.nlm.nih.gov/pubmed). The pre- protein sequences in Homo sapiens (PubMed protein database, viously detected phosphosites with unknown biological function www.ncbi.nlm.nih.gov/protein)andBLAST(http://blast.ncbi.nlm. are not labeled. LC-MS/MS data were analyzed for kinase– nih.gov/Blast.cgi) search of the following species: Homo sapiens, substrates using NetPhorest (7) (http://netphorest.info), for Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, kinase–substrate network reconstruction using NetworKIN (8) Arabidopsis thaliana, and Saccharomyces cerevisiae. 1. Villén J, Gygi SP (2008) The SCX/IMAC enrichment approach for global phosphorylation 9. Hernandez M, Lachmann A, Zhao S, Xiao K, Ma’ayan A (2010) Inferring the sign of analysis by mass spectrometry.
Load more

Recommended publications
  • Environmental Influences on Endothelial Gene Expression
    ENDOTHELIAL CELL GENE EXPRESSION John Matthew Jeff Herbert Supervisors: Prof. Roy Bicknell and Dr. Victoria Heath PhD thesis University of Birmingham August 2012 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. ABSTRACT Tumour angiogenesis is a vital process in the pathology of tumour development and metastasis. Targeting markers of tumour endothelium provide a means of targeted destruction of a tumours oxygen and nutrient supply via destruction of tumour vasculature, which in turn ultimately leads to beneficial consequences to patients. Although current anti -angiogenic and vascular targeting strategies help patients, more potently in combination with chemo therapy, there is still a need for more tumour endothelial marker discoveries as current treatments have cardiovascular and other side effects. For the first time, the analyses of in-vivo biotinylation of an embryonic system is performed to obtain putative vascular targets. Also for the first time, deep sequencing is applied to freshly isolated tumour and normal endothelial cells from lung, colon and bladder tissues for the identification of pan-vascular-targets. Integration of the proteomic, deep sequencing, public cDNA libraries and microarrays, delivers 5,892 putative vascular targets to the science community.
    [Show full text]
  • Transcriptomic Analysis of Native Versus Cultured Human and Mouse Dorsal Root Ganglia Focused on Pharmacological Targets Short
    bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Transcriptomic analysis of native versus cultured human and mouse dorsal root ganglia focused on pharmacological targets Short title: Comparative transcriptomics of acutely dissected versus cultured DRGs Andi Wangzhou1, Lisa A. McIlvried2, Candler Paige1, Paulino Barragan-Iglesias1, Carolyn A. Guzman1, Gregory Dussor1, Pradipta R. Ray1,#, Robert W. Gereau IV2, # and Theodore J. Price1, # 1The University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, 800 W Campbell Rd. Richardson, TX, 75080, USA 2Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine # corresponding authors [email protected], [email protected] and [email protected] Funding: NIH grants T32DA007261 (LM); NS065926 and NS102161 (TJP); NS106953 and NS042595 (RWG). The authors declare no conflicts of interest Author Contributions Conceived of the Project: PRR, RWG IV and TJP Performed Experiments: AW, LAM, CP, PB-I Supervised Experiments: GD, RWG IV, TJP Analyzed Data: AW, LAM, CP, CAG, PRR Supervised Bioinformatics Analysis: PRR Drew Figures: AW, PRR Wrote and Edited Manuscript: AW, LAM, CP, GD, PRR, RWG IV, TJP All authors approved the final version of the manuscript. 1 bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Supplementary Table 1
    SI Table S1. Broad protein kinase selectivity for PF-2771. Kinase, PF-2771 % Inhibition at 10 μM Service Kinase, PF-2771 % Inhibition at 1 μM Service rat RPS6KA1 (RSK1) 39 Dundee AURKA (AURA) 24 Invitrogen IKBKB (IKKb) 26 Dundee CDK2 /CyclinA 21 Invitrogen mouse LCK 25 Dundee rabbit MAP2K1 (MEK1) 19 Dundee AKT1 (AKT) 21 Dundee IKBKB (IKKb) 16 Dundee CAMK1 (CaMK1a) 19 Dundee PKN2 (PRK2) 14 Dundee RPS6KA5 (MSK1) 18 Dundee MAPKAPK5 14 Dundee PRKD1 (PKD1) 13 Dundee PIM3 12 Dundee MKNK2 (MNK2) 12 Dundee PRKD1 (PKD1) 12 Dundee MARK3 10 Dundee NTRK1 (TRKA) 12 Invitrogen SRPK1 9 Dundee MAPK12 (p38g) 11 Dundee MAPKAPK5 9 Dundee MAPK8 (JNK1a) 11 Dundee MAPK13 (p38d) 8 Dundee rat PRKAA2 (AMPKa2) 11 Dundee AURKB (AURB) 5 Dundee NEK2 11 Invitrogen CSK 5 Dundee CHEK2 (CHK2) 11 Invitrogen EEF2K (EEF-2 kinase) 4 Dundee MAPK9 (JNK2) 9 Dundee PRKCA (PKCa) 4 Dundee rat RPS6KA1 (RSK1) 8 Dundee rat PRKAA2 (AMPKa2) 4 Dundee DYRK2 7 Dundee rat CSNK1D (CKId) 3 Dundee AKT1 (AKT) 7 Dundee LYN 3 BioPrint PIM2 7 Invitrogen CSNK2A1 (CKIIa) 3 Dundee MAPK15 (ERK7) 6 Dundee CAMKK2 (CAMKKB) 1 Dundee mouse LCK 5 Dundee PIM3 1 Dundee PDPK1 (PDK1) (directed 5 Invitrogen rat DYRK1A (MNB) 1 Dundee RPS6KB1 (p70S6K) 5 Dundee PBK 0 Dundee CSNK2A1 (CKIIa) 4 Dundee PIM1 -1 Dundee CAMKK2 (CAMKKB) 4 Dundee DYRK2 -2 Dundee SRC 4 Invitrogen MAPK12 (p38g) -2 Dundee MYLK2 (MLCK_sk) 3 Invitrogen NEK6 -3 Dundee MKNK2 (MNK2) 2 Dundee RPS6KB1 (p70S6K) -3 Dundee SRPK1 2 Dundee AKT2 -3 Dundee MKNK1 (MNK1) 2 Dundee RPS6KA3 (RSK2) -3 Dundee CHEK1 (CHK1) 2 Invitrogen rabbit MAP2K1 (MEK1) -4 Dundee
    [Show full text]
  • UPSTREAM COMPONENTS of MTORC1 by Li Li a Dissertation
    UPSTREAM COMPONENTS OF MTORC1 by Li Li A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biological Chemistry) in The University of Michigan 2011 Doctoral Committee: Professor Kunliang Guan, Chair Professor Randal J. Kaufman Associate Professor Anne B. Vojtek Associate Professor Martin G. Myers Assistant Professor Diane C. Fingar © Li Li 2011 This work is dedicated to my parents. Without their support and guidance, I would not be where I am today. I also dedicate this work to my husband, for pushing me to the best I can. Last, and most importantly, I would like to dedicate this work to my fifteen months son for being well behaved during the last few months. You are the treasure of my life. ii ACKNOWLEDGEMENTS I would like to express my deep gratitude to my mentor Dr. Kun-Liang Guan for his direction, encouragement, and support during my graduate study. I am very thankful to him for his patience with me, enduring my naïve questions. That Dr. Guan is so approachable and enthusiastic about science set me an example on how to be a great PI and a great scientist. Next, I sincerely acknowledge the members of my thesis committee, Drs Randal J. Kaufman, Anne B. Vojtek, Diane C. Fingar and Martin G. Myers for their insightful suggestions and constructive criticism on my work. I would like to thank the current and former members of the Guan lab. All of them made the lab a great place to work. Especially, I would like to thank Bin Zhao, my husband, also a Guan lab member, for teaching me every lab techniques.
    [Show full text]
  • Supplementary Table 1. in Vitro Side Effect Profiling Study for LDN/OSU-0212320. Neurotransmitter Related Steroids
    Supplementary Table 1. In vitro side effect profiling study for LDN/OSU-0212320. Percent Inhibition Receptor 10 µM Neurotransmitter Related Adenosine, Non-selective 7.29% Adrenergic, Alpha 1, Non-selective 24.98% Adrenergic, Alpha 2, Non-selective 27.18% Adrenergic, Beta, Non-selective -20.94% Dopamine Transporter 8.69% Dopamine, D1 (h) 8.48% Dopamine, D2s (h) 4.06% GABA A, Agonist Site -16.15% GABA A, BDZ, alpha 1 site 12.73% GABA-B 13.60% Glutamate, AMPA Site (Ionotropic) 12.06% Glutamate, Kainate Site (Ionotropic) -1.03% Glutamate, NMDA Agonist Site (Ionotropic) 0.12% Glutamate, NMDA, Glycine (Stry-insens Site) 9.84% (Ionotropic) Glycine, Strychnine-sensitive 0.99% Histamine, H1 -5.54% Histamine, H2 16.54% Histamine, H3 4.80% Melatonin, Non-selective -5.54% Muscarinic, M1 (hr) -1.88% Muscarinic, M2 (h) 0.82% Muscarinic, Non-selective, Central 29.04% Muscarinic, Non-selective, Peripheral 0.29% Nicotinic, Neuronal (-BnTx insensitive) 7.85% Norepinephrine Transporter 2.87% Opioid, Non-selective -0.09% Opioid, Orphanin, ORL1 (h) 11.55% Serotonin Transporter -3.02% Serotonin, Non-selective 26.33% Sigma, Non-Selective 10.19% Steroids Estrogen 11.16% 1 Percent Inhibition Receptor 10 µM Testosterone (cytosolic) (h) 12.50% Ion Channels Calcium Channel, Type L (Dihydropyridine Site) 43.18% Calcium Channel, Type N 4.15% Potassium Channel, ATP-Sensitive -4.05% Potassium Channel, Ca2+ Act., VI 17.80% Potassium Channel, I(Kr) (hERG) (h) -6.44% Sodium, Site 2 -0.39% Second Messengers Nitric Oxide, NOS (Neuronal-Binding) -17.09% Prostaglandins Leukotriene,
    [Show full text]
  • 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.
    [Show full text]
  • Analysis of 3-Phosphoinositide-Dependent Kinase-1 Signaling and Function in ES Cells
    EXPERIMENTAL CELL RESEARCH 314 (2008) 2299– 2312 available at www.sciencedirect.com www.elsevier.com/locate/yexcr Research Article Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells Tanja Tamgüneya,b, Chao Zhangc, Dorothea Fiedlerc, Kevan Shokatc, David Stokoea,⁎ aUCSF Cancer Research Institute, USA bMolecular Medicine Program, Friedrich-Alexander University of Erlangen-Nuremberg, Erlangen, Germany cDepartment of Cellular and Molecular Pharmacology, University of California, San Francisco, 2340 Sutter Street, San Francisco, CA 94115, USA ARTICLE INFORMATION ABSTRACT Article Chronology: 3-Phosphoinositide-dependent kinase-1 (PDK1) phosphorylates and activates several Received 5 February 2008 kinases in the cAMP-dependent, cGMP-dependent and protein kinase C (AGC) family. Revised version received Many putative PDK1 substrates have been identified, but have not been analyzed following 15 April 2008 transient and specific inhibition of PDK1 activity. Here, we demonstrate that a previously Accepted 16 April 2008 characterized PDK1 inhibitor, BX-795, shows biological effects that are not consistent with Available online 23 April 2008 PDK1 inhibition. Therefore, we describe the creation and characterization of a PDK1 mutant, L159G, which can bind inhibitor analogues containing bulky groups that hinder access to the − − Keywords: ATP binding pocket of wild type (WT) kinases. When expressed in PDK1 / ES cells, PDK1 PDK1 L159G restored phosphorylation of PDK1 targets known to be hypophosphorylated in these PKB/Akt cells. Screening of multiple inhibitor analogues showed that 1-NM-PP1 and 3,4-DMB-PP1 − − PI3K optimally inhibited the phosphorylation of PDK1 targets in PDK1 / ES cells expressing PDK1 Chemical genetics L159G but not WT PDK1. These compounds confirmed previously assumed PDK1 substrates, AGC kinases but revealed distinct dephosphorylation kinetics.
    [Show full text]
  • Development and Validation of a Protein-Based Risk Score for Cardiovascular Outcomes Among Patients with Stable Coronary Heart Disease
    Supplementary Online Content Ganz P, Heidecker B, Hveem K, et al. Development and validation of a protein-based risk score for cardiovascular outcomes among patients with stable coronary heart disease. JAMA. doi: 10.1001/jama.2016.5951 eTable 1. List of 1130 Proteins Measured by Somalogic’s Modified Aptamer-Based Proteomic Assay eTable 2. Coefficients for Weibull Recalibration Model Applied to 9-Protein Model eFigure 1. Median Protein Levels in Derivation and Validation Cohort eTable 3. Coefficients for the Recalibration Model Applied to Refit Framingham eFigure 2. Calibration Plots for the Refit Framingham Model eTable 4. List of 200 Proteins Associated With the Risk of MI, Stroke, Heart Failure, and Death eFigure 3. Hazard Ratios of Lasso Selected Proteins for Primary End Point of MI, Stroke, Heart Failure, and Death eFigure 4. 9-Protein Prognostic Model Hazard Ratios Adjusted for Framingham Variables eFigure 5. 9-Protein Risk Scores by Event Type This supplementary material has been provided by the authors to give readers additional information about their work. Downloaded From: https://jamanetwork.com/ on 10/02/2021 Supplemental Material Table of Contents 1 Study Design and Data Processing ......................................................................................................... 3 2 Table of 1130 Proteins Measured .......................................................................................................... 4 3 Variable Selection and Statistical Modeling ........................................................................................
    [Show full text]
  • Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications
    Stem Cell Rev and Rep DOI 10.1007/s12015-016-9662-8 Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications Behnam Ahmadian Baghbaderani 1 & Adhikarla Syama2 & Renuka Sivapatham3 & Ying Pei4 & Odity Mukherjee2 & Thomas Fellner1 & Xianmin Zeng3,4 & Mahendra S. Rao5,6 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract We have recently described manufacturing of hu- help determine which set of tests will be most useful in mon- man induced pluripotent stem cells (iPSC) master cell banks itoring the cells and establishing criteria for discarding a line. (MCB) generated by a clinically compliant process using cord blood as a starting material (Baghbaderani et al. in Stem Cell Keywords Induced pluripotent stem cells . Embryonic stem Reports, 5(4), 647–659, 2015). In this manuscript, we de- cells . Manufacturing . cGMP . Consent . Markers scribe the detailed characterization of the two iPSC clones generated using this process, including whole genome se- quencing (WGS), microarray, and comparative genomic hy- Introduction bridization (aCGH) single nucleotide polymorphism (SNP) analysis. We compare their profiles with a proposed calibra- Induced pluripotent stem cells (iPSCs) are akin to embryonic tion material and with a reporter subclone and lines made by a stem cells (ESC) [2] in their developmental potential, but dif- similar process from different donors. We believe that iPSCs fer from ESC in the starting cell used and the requirement of a are likely to be used to make multiple clinical products. We set of proteins to induce pluripotency [3]. Although function- further believe that the lines used as input material will be used ally identical, iPSCs may differ from ESC in subtle ways, at different sites and, given their immortal status, will be used including in their epigenetic profile, exposure to the environ- for many years or even decades.
    [Show full text]
  • CCND1 and DUSP9 Are Promising Druggable Targets for Treating
    Sequence and Pathway analysis CCND1 and DUSP9 are promising druggable targets for treating Neoplasm Metastasis and Osteosarcoma that control activity of EGR1, EP300 and RXRA transcription factors on promoters of differentially expressed genes Demo User geneXplain GmbH [email protected] Data received on 07/09/2019 ; Run on 17/06/2020 ; Report generated on 17/06/2020 Genome Enhancer release 2.0 (TRANSFAC®, TRANSPATH® and HumanPSD™ release 2020.2) Abstract In the present study we applied the software package "Genome Enhancer" to a multiomics data set that contains transcriptomics and proteomics data. The study is done in the context of Neoplasm Metastasis and Osteosarcoma. The goal of this pipeline is to identify potential drug targets in the molecular network that governs the studied pathological process. In the first step of analysis pipeline discovers transcription factors (TFs) that regulate genes activities in the pathological state. The activities of these TFs are controlled by so-called master regulators, which are identified in the second step of analysis. After a subsequent druggability checkup, the most promising master regulators are chosen as potential drug targets for the analyzed pathology. At the end the pipeline comes up with (a) a list of known drugs and (b) investigational active chemical compounds with the potential to interact with selected drug targets. From the data set analyzed in this study, we found the following TFs to be potentially involved in the regulation of the differentially expressed genes: EGR1, EP300, NR3C1, RXRA, SMAD2 and SMAD1. The subsequent network analysis suggested integrins SIRT1 Cdk4-isoform1:cyclinD1a TFIIH-CAK MKP-4 as the most promising molecular targets for further research, drug development and drug repurposing initiatives on the basis of identified molecular mechanism of the studied pathology.
    [Show full text]
  • Defining Endothelial Cell Functional Heterogeneity and Plasticity Using Single-Cell RNA-Sequencing
    Defining endothelial cell functional heterogeneity and plasticity using single-cell RNA-sequencing The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Kalluri, Aditya Sreemadhav. 2019. Defining endothelial cell functional heterogeneity and plasticity using single-cell RNA- sequencing. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42013060 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 Title Pag e Defining endothelial cell functional heterogeneity and plasticity using single-cell RNA-sequencing A dissertation presented by Aditya Sreemadhav Kalluri to The Committee on Higher Degrees in Biophysics in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biophysics Harvard University Cambridge, Massachusetts June 2019 i © 2019 – Aditya Sreemadhav Kalluri All rights reserved. Cop yrig ht ii Dissertation Advisor: Professor Elazer R. Edelman Aditya Sreemadhav Kalluri Defining endothelial cell functional heterogeneity and plasticity using single-cell RNA-sequencing Abstract The state of the endothelium defines vascular health – ensuring homeostasis when intact and driving pathology when dysfunctional. The endothelial cells (ECs) which comprise the endothelium are necessarily highly responsive and reflect their local mechanical, biological, and cellular context, making a concrete definition of endothelial state elusive. Heterogeneity is the hallmark of ECs and has long been studied in the context of structural and functional differences between organs and vascular beds.
    [Show full text]
  • Activation of Diverse Signalling Pathways by Oncogenic PIK3CA Mutations
    ARTICLE Received 14 Feb 2014 | Accepted 12 Aug 2014 | Published 23 Sep 2014 DOI: 10.1038/ncomms5961 Activation of diverse signalling pathways by oncogenic PIK3CA mutations Xinyan Wu1, Santosh Renuse2,3, Nandini A. Sahasrabuddhe2,4, Muhammad Saddiq Zahari1, Raghothama Chaerkady1, Min-Sik Kim1, Raja S. Nirujogi2, Morassa Mohseni1, Praveen Kumar2,4, Rajesh Raju2, Jun Zhong1, Jian Yang5, Johnathan Neiswinger6, Jun-Seop Jeong6, Robert Newman6, Maureen A. Powers7, Babu Lal Somani2, Edward Gabrielson8, Saraswati Sukumar9, Vered Stearns9, Jiang Qian10, Heng Zhu6, Bert Vogelstein5, Ben Ho Park9 & Akhilesh Pandey1,8,9 The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets. 1 McKusick-Nathans Institute of Genetic Medicine and Department of Biological Chemistry, Johns Hopkins University School of Medicine, 733 North Broadway, BRB 527, Baltimore, Maryland 21205, USA.
    [Show full text]