DOCSLIB.ORG

Caspase-3-Mediated Cleavage of Rad9 During Apoptosis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Caspase-3-Mediated Cleavage of Rad9 During Apoptosis Oncogene (2003) 22, 6340–6346 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Caspase-3-mediated cleavage of Rad9 during apoptosis Michael W Lee1,2, Itaru Hirai1,3 and Hong-Gang Wang*,1,2,3 1Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, USA; 2Department of Pharmacology & Therapeutics, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612, USA; 3Department of Interdisciplinary Oncology, University of South Florida College of Medicine, 12902 Magnolia Drive, Tampa, FL 33612, USA The activation of caspases is a critical event for the distinct from necrosis.Grossly, apoptotic cells exhibit a execution phase of programmed cell death. Caspases are variety of identifiable characteristics such as membrane highly specific in their ability to activate or inhibit many blebbing, chromatin condensation, and typically, crucial proteins in the cell via cleavage. In this study, we fragmentation into membrane-enclosed vesicles report the identification of several caspase-3-like cleavage (Earnshaw et al., 1999). Biochemical changes include sites in the cell-cycle checkpoint protein Rad9. We the externalization of phosphatidylserine, degradation demonstrate that human Rad9 can be specifically cleaved of chromosomal DNA into high-molecular-weight in cells induced to enter apoptosis by both DNA damage oligonucleosomal fragments, and cleavage of specific and staurosporine treatment. Indeed, we show that human polypeptides such as PARP, ICAD, and antiapoptotic Rad9 can be effectively cleaved both in vitro and in vivo, proteins such as Bcl-2 and Bcl-XL. which can be inhibited by either a pan-caspase inhibitor or Many stimuli that trigger apoptosis without ligating a caspase-3-specific inhibitor. Additionally, no cleavage death receptors cause mitochondria to release the of Rad9 can be seen in the caspase-3-deficient cell line electron transport protein cytochrome c to the cytosol MCF-7. Site-directed mutagenesis of three of the most (Green and Reed, 1998).Apaf-1 binds the released conserved cleavage sites dramatically abrogates cleavage cytochrome c, and then undergoes a nucleotide-depen- of Rad9 by caspase-3 in vitro, and in intact cells after dent conformational change that allows the binding of DNA damage. Expression of the cleavage-resistant procaspase-9.As a consequence of this binding, mutant Rad9 DDD/AAA appears to protect the cell from procaspase-9 is cleaved to active caspase-9, which in DNA damage-induced apoptosis. Immunofluorescence turn leads to activation of the downstream effector studies of Rad9 localization before and after induction caspase-3, -6, and -7.These effector caspases then of apoptosis showa translocation of Rad9 from the directly cause the morphological changes associated nucleus to the cytosol, concomitant to the appearance of with apoptosis by specifically cleaving ‘death substrates’ apoptotic morphology. Furthermore, analysis of a trun- (Earnshaw et al., 1999). Caspases cleave a limited cated Rad9 mutant that corresponds to a putative N- number of cellular proteins, and the process is one of terminal cleavage fragment shows that the N-terminal limited proteolysis (Salvesen and Dixit, 1997).Owing to portion of Rad9 localizes in the cytosol, binds to Bcl-XL, their substrate specificity, caspase-mediated cleavage and induces apoptosis. These results support a dual role can result in activation or inactivation of their protein for cleavage of Rad9: (1) the liberation and translocation substrates, but never degradation (Salvesen and Dixit, of the BH3 domain-containing N-terminus of Rad9 to the 1997). cytosol, as a means of promoting apoptosis via antagon- The Bcl-2 family is comprised of well over a dozen ism of Bcl-XL, and (2) the disruption of the Rad9-Rad1- proteins, which have been classified into three functional Hus1 DNA damage checkpoint complex. groups (Wang and Reed, 1998).Members of the first Oncogene (2003) 22, 6340–6346. doi:10.1038/sj.onc.1206729 group such as Bcl-2 and Bcl-XL contain four conserved Bcl-2 homology (BH) domains (BH1–BH4), and possess Keywords: caspase-3; Rad9; DNA damage; cell-cycle antiapoptotic activity.In contrast, group II consists of checkpoint; apoptosis proapoptotic Bcl-2 family members including Bax and Bak, which have a similar overall structure to group I proteins but lack the N-terminal BH4 domain.Group III consists of a large and diverse collection of Introduction proapoptotic proteins whose only common feature is the presence of the BH3 domain.The Bcl-2 family Apoptosis is an evolutionarily conserved process of cell of proteins appears to control the ‘decision’ step of suicide that is both morphologically and biochemically apoptosis, determining whether certain caspases will or will not become activated (Reed, 1998).Antiapoptotic *Correspondence: H-G Wang, Drug Discovery Program, H.Lee members of the Bcl-2 family tend to prevent activation Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, of these terminal effector proteases by blocking the Tampa FL 33612, USA; E-mail: wanghg@moffitt.usf.edu Received 13 February 2003; revised 17 April 2003; accepted 22 April release of apoptogenic molecules such as cytochrome c 2003 from the mitochondria.Conversely, proapoptotic Caspase-3 cleavage of Rad9 MW Lee et al 6341 Bcl-2 family members facilitate caspase activation by promoting the release of cytochrome c from the mitochondria. In response to DNA damage, which can arise spontaneously from replication errors or from exogen- ous agents, a rapid-surveillance signaling cascade called the DNA damage checkpoint is initiated (Caspari and Carr, 2002).Activation of this cascade results in cell- cycle arrest, which provides time for the damage to be repaired.Deregulation of this checkpoint can lead to genomic instability that is associated with genetic diseases such as cancer (Hartwell and Kastan, 1994). The Rad family of checkpoint proteins have emerged not only as critical mediators of this damage response pathway but also as putative apical sensors in the DNA damage checkpoint.Indeed, three members of the Rad Figure 1 Human Rad9 is degraded after treatment with high-dose IR.U937 cells were treated with various doses of IR and cultured family Rad9, Rad1, and Hus1 have been shown to for 5 h after treatment.Lysate was prepared and subjected to interact with each other in a manner similar to the SDS—PAGE, and transferred to nitrocellulose filters for analysis of proliferating cell nuclear antigen (PCNA) (O’Connell Rad9 expression using a polyclonal anti-Rad9 antibody (top).The et al., 2000). Furthermore, a fourth Rad family member, same blot was then stripped and reprobed with anti-caspase-3 Rad17, bears structural similarity to replication factor C antibody (bottom) (RFC), the clamp loader which places PCNA onto DNA (Tsurimoto, 1999; Venclovas and Thelen, 2000).It et al., 2000b; St Onge et al., 2001), when resolved on is believed that, in times of damage, these four proteins SDS–polyacrylamide gel (SDS–PAGE), Rad9 migrates form a putative DNA damage sensor, which encircles as several distinct species ranging from 42 to 60 kDa damaged DNA and relays the damage signal to down- even in the absence of genotoxic stress.These different stream effectors (Zhou and Elledge, 2000; Wahl and bands are likely the result of phosphorylation, and can Carr, 2001).In addition, it was shown that Rad9 could be seen in both treated and untreated cells (St.Onge interact with Bcl-2 and Bcl-XL via a conserved BH3 et al., 1999, 2001; Volkmer and Karnitz, 1999; Komatsu domain located in the N-terminus of the Rad9 protein et al., 2000b; Chen et al., 2001). As shown in Figure 1, to antagonize the protective function of these proteins, for example, Rad9 appeared as three major species, promoting apoptosis (Komatsu et al., 2000a, b; Yoshida designated Rad9a, Rad9b, and Rad9g, on Western et al., 2002). It has also been demonstrated that Rad9 blots.Interestingly, after treating U937 cells with can be phosphorylated on Tyr-28 within the BH3 incrementally increasing doses of ionizing radiation domain by c-Abl, and that this phosphorylation (IR), we observed that the lesser modified forms (b, g) promotes the interaction of Rad9 with Bcl-XL (Yoshida of Rad9 decreased in a dose-dependent manner et al., 2002). (Figure 1).This decrease does not directly translate into In this study, we demonstrate that Rad9 possess the increases in the highly modified form (Rad9a), several potential caspase-3 cleavage sites and can be suggesting that Rad9, especially the lesser modified cleaved by caspase-3 both in vitro and in vivo.After forms (Rad9b and Rad9g), might undergo proteolysis exposure to DNA-damaging agents or staurosporine after DNA damage.Reprobing the same blot with anti- (STS), Rad9 undergoes proteolysis and cytosolic trans- caspase-3 antibody revealed that the decrease in Rad9 location, which can be blocked by caspase inhibitors. protein level was concomitant to the activation of Mutating the caspase-3 cleavage sites in Rad9 blocks caspase-3 (Figure 1).Upon analysis of the Rad9 protein cleavage in vivo, and also appears to protect the cell sequence, we noticed that human Rad9 contains several from death induced by DNA-damaging agents.Further- potential caspase-3 cleavage sites (Figure 2a).For more, Rad9DC, a mutant Rad9 that lacks the C- example, the sequence DDID (amino acids 301–304) is terminus and thus mimics a potential N-terminal quite similar to the consensus motif DXXD for caspase- cleavage fragment of Rad9, retains the
Load more

Recommended publications
  • 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]
  • Hypermethylation of RAD9A Intron 2 in Childhood Cancer Patients, Leukemia and Tumor Cell Lines Suggest a Role for Oncogenic Transformation
    Hypermethylation of RAD9A intron 2 in childhood cancer patients, leukemia and tumor cell lines suggest a role for oncogenic transformation Danuta Galetzka ( [email protected] ) Johannes Gutenberg Universitat Mainz https://orcid.org/0000-0003-1825-9136 Julia Boeck Institute of Human Genetics, Julius Maximilians University Würzburg Marcus Dittrich Bioinformatics Department Julius-Maximilians-Universitat Wurzburg Olesja Sinizyn Department of Radiation Oncology and Radiations Therapy , University Medical Centre, Mainz Marco Ludwig DRK Medical Centre Alzey Heidi Rossmann Institute of Clinical Chemistry and Laboratory Medicine, University Medical Centre, Mainz Claudia Spix German Childhood Cancer Registry, Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Centre, Mainz Markus Radsak Departement of Hematology, University Medical Centre, Mainz Peter Scholz-Kreisel Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Centre, Mainz Johanna Mirsch Radiation Biology and DNA Repair, Technical University, Darmstadt Matthias Linke Institute of Human Genetics, University Medical Centre, Mainz Walburgis Brenner Departement of Obsterics and Womans Health, University Medical Centre, Mainz Manuela Marron Leibniz Institute for Prevention Research and Epidemiology, BIPS, Bremen Alicia Poplawski Institute of Medical Biostatistics, Epidemiology and Informatics, University Medical Centre, Mainz Dirk Prawitt Center for Pediatrics and Adolescent Medicine, University Medical Centre, Mainz Thomas Haaf Institute of Human Genetics, Julius Maximilians University, Würzburg Heinz Schmidberger Department of Radiation Oncology and Radiation Therapy, University Centre, Mainz Research article Keywords: RAD9A, childhood cancer, hypermethylation, normal body cells, somatic mosaicism, leukemia Posted Date: August 12th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-55470/v1 Page 1/22 License: This work is licensed under a Creative Commons Attribution 4.0 International License.
    [Show full text]
  • HUS1 Regulates in Vivo Responses to Genotoxic Chemotherapies
    Oncogene (2016) 35, 662–669 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc SHORT COMMUNICATION HUS1 regulates in vivo responses to genotoxic chemotherapies G Balmus1, PX Lim1, A Oswald1, KR Hume1,2, A Cassano1, J Pierre1, A Hill1, W Huang3, A August3, T Stokol4, T Southard1 and RS Weiss1 Cells are under constant attack from genotoxins and rely on a multifaceted DNA damage response (DDR) network to maintain genomic integrity. Central to the DDR are the ATM and ATR kinases, which respond primarily to double-strand DNA breaks (DSBs) and replication stress, respectively. Optimal ATR signaling requires the RAD9A-RAD1-HUS1 (9-1-1) complex, a toroidal clamp that is loaded at damage sites and scaffolds signaling and repair factors. Whereas complete ATR pathway inactivation causes embryonic lethality, partial Hus1 impairment has been accomplished in adult mice using hypomorphic (Hus1neo) and null (Hus1Δ1) Hus1 alleles, and here we use this system to define the tissue- and cell type-specific actions of the HUS1-mediated DDR in vivo. Hus1neo/Δ1 mice showed hypersensitivity to agents that cause replication stress, including the crosslinking agent mitomycin C (MMC) and the replication inhibitor hydroxyurea, but not the DSB inducer ionizing radiation. Analysis of tissue morphology, genomic instability, cell proliferation and apoptosis revealed that MMC treatment caused severe damage in highly replicating tissues of mice with partial Hus1 inactivation. The role of the 9-1-1 complex in responding to MMC was partially ATR-independent, as a HUS1 mutant that was proficient for ATR-induced checkpoint kinase 1 phosphorylation nevertheless conferred MMC hypersensitivity.
    [Show full text]
  • Rabbit Anti-Phospho-C-Abl-SL4086R-FITC
    SunLong Biotech Co.,LTD Tel: 0086-571- 56623320 Fax:0086-571- 56623318 E-mail:[email protected] www.sunlongbiotech.com Rabbit Anti-phospho-c-Abl SL4086R-FITC Product Name: Anti-phospho-c-Abl(Tyr276)/FITC Chinese Name: FITC标记的磷酸化非受体酪氨酸激酶c-Abl抗体 Abelson Murine Leukemia Viral Oncogene Homolog 1; Abelson murine leukemia viral v abl oncogene homolog 1; Abl 1; ABL; Abl protein; Abl1; Bcr/c abl oncogene protein; Alias: JTK 7; JTK7; p150 ; Proto oncogene tyrosine protein kinase ABL1; Transformation gene oncogene ABL; v abl Abelson murine leukemia viral oncogene homolog 1; v abl. Organism Species: Rabbit Clonality: Polyclonal React Species: Human,Mouse,Rat,Dog,Pig,Cow,Horse,Rabbit, IF=1:50-200 Applications: not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. Molecular weight: 126kDa Cellular localization: The cell membrane Form: Lyophilized or Liquid Concentration: 1mg/ml KLHwww.sunlongbiotech.com conjugated Synthesised phosphopeptide derived from human c-Abl isoform b immunogen: around the phosphorylation site of Tyr276 Lsotype: IgG Purification: affinity purified by Protein A Storage Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year Storage: when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. background: The c Abl proto oncogene encodes a protein tyrosine kinase that is located in the Product Detail: cytoplasm and nucleus.
    [Show full text]
  • RAD9A Rabbit Pab
    Leader in Biomolecular Solutions for Life Science RAD9A Rabbit pAb Catalog No.: A1890 Basic Information Background Catalog No. This gene product is highly similar to Schizosaccharomyces pombe rad9, a cell cycle A1890 checkpoint protein required for cell cycle arrest and DNA damage repair. This protein possesses 3' to 5' exonuclease activity, which may contribute to its role in sensing and Observed MW repairing DNA damage. It forms a checkpoint protein complex with RAD1 and HUS1. This 60kDa complex is recruited by checkpoint protein RAD17 to the sites of DNA damage, which is thought to be important for triggering the checkpoint-signaling cascade. Alternatively Calculated MW spliced transcript variants encoding different isoforms have been found for this gene. 42kDa Category Primary antibody Applications WB, IHC, IF Cross-Reactivity Human, Rat Recommended Dilutions Immunogen Information WB 1:500 - 1:2000 Gene ID Swiss Prot 5883 Q99638 IHC 1:50 - 1:200 Immunogen 1:50 - 1:200 IF Recombinant fusion protein containing a sequence corresponding to amino acids 162-391 of human RAD9A (NP_004575.1). Synonyms RAD9A;RAD9 Contact Product Information www.abclonal.com Source Isotype Purification Rabbit IgG Affinity purification Storage Store at -20℃. Avoid freeze / thaw cycles. Buffer: PBS with 0.02% sodium azide,50% glycerol,pH7.3. Validation Data Western blot analysis of extracts of various cell lines, using RAD9A antibody (A1890) at 1:1000 dilution. Secondary antibody: HRP Goat Anti-Rabbit IgG (H+L) (AS014) at 1:10000 dilution. Lysates/proteins: 25ug per lane. Blocking buffer: 3% nonfat dry milk in TBST. Detection: ECL Basic Kit (RM00020). Immunohistochemistry of paraffin- Immunohistochemistry of paraffin- Immunofluorescence analysis of MCF-7 embedded rat liver using RAD9A antibody embedded human liver damage using cells using RAD9A antibody (A1890).
    [Show full text]
  • Differential Expression Profile Analysis of DNA Damage Repair Genes in CD133+/CD133‑ Colorectal Cancer Cells
    ONCOLOGY LETTERS 14: 2359-2368, 2017 Differential expression profile analysis of DNA damage repair genes in CD133+/CD133‑ colorectal cancer cells YUHONG LU1*, XIN ZHOU2*, QINGLIANG ZENG2, DAISHUN LIU3 and CHANGWU YUE3 1College of Basic Medicine, Zunyi Medical University, Zunyi; 2Deparment of Gastroenterological Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi;3 Zunyi Key Laboratory of Genetic Diagnosis and Targeted Drug Therapy, The First People's Hospital of Zunyi, Zunyi, Guizhou 563003, P.R. China Received July 20, 2015; Accepted January 6, 2017 DOI: 10.3892/ol.2017.6415 Abstract. The present study examined differential expression cells. By contrast, 6 genes were downregulated and none levels of DNA damage repair genes in COLO 205 colorectal were upregulated in the CD133+ cells compared with the cancer cells, with the aim of identifying novel biomarkers for COLO 205 cells. These findings suggest that CD133+ cells the molecular diagnosis and treatment of colorectal cancer. may possess the same DNA repair capacity as COLO 205 COLO 205-derived cell spheres were cultured in serum-free cells. Heterogeneity in the expression profile of DNA damage medium supplemented with cell factors, and CD133+/CD133- repair genes was observed in COLO 205 cells, and COLO cells were subsequently sorted using an indirect CD133 205-derived CD133- cells and CD133+ cells may therefore microbead kit. In vitro differentiation and tumorigenicity assays provide a reference for molecular diagnosis, therapeutic target in BABA/c nude mice were performed to determine whether selection and determination of the treatment and prognosis for the CD133+ cells also possessed stem cell characteristics, in colorectal cancer.
    [Show full text]
  • Alterations of the Interactome of Bcl-2 Proteins in Breast Cancer at the Transcriptional, Mutational and Structural Level
    RESEARCH ARTICLE Alterations of the interactome of Bcl-2 proteins in breast cancer at the transcriptional, mutational and structural level Simon Mathis Kønig1, Vendela Rissler1, Thilde Terkelsen1, Matteo Lambrughi1, 1,2 Elena PapaleoID * 1 Computational Biology Laboratory, Danish Cancer Society Research Center, Copenhagen, Denmark, a1111111111 2 Translational Disease Systems Biology, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research University of Copenhagen, Copenhagen, Denmark a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 Abstract Apoptosis is an essential defensive mechanism against tumorigenesis. Proteins of the B- OPEN ACCESS cell lymphoma-2 (Bcl-2) family regulate programmed cell death by the mitochondrial apopto- sis pathway. In response to intracellular stress, the apoptotic balance is governed by inter- Citation: Kønig SM, Rissler V, Terkelsen T, Lambrughi M, Papaleo E (2019) Alterations of the actions of three distinct subgroups of proteins; the activator/sensitizer BH3 (Bcl-2 homology interactome of Bcl-2 proteins in breast cancer at 3)-only proteins, the pro-survival, and the pro-apoptotic executioner proteins. Changes in the transcriptional, mutational and structural level. expression levels, stability, and functional impairment of pro-survival proteins can lead to an PLoS Comput Biol 15(12): e1007485. https://doi. imbalance in tissue homeostasis. Their overexpression or hyperactivation can result in org/10.1371/journal.pcbi.1007485 oncogenic effects. Pro-survival Bcl-2 family members carry out their function by binding the Editor: Igor N. Berezovsky, A�STAR Singapore, BH3 short linear motif of pro-apoptotic proteins in a modular way, creating a complex net- SINGAPORE work of protein-protein interactions. Their dysfunction enables cancer cells to evade cell Received: July 8, 2019 death.
    [Show full text]
  • Epigenetic Mechanisms Are Involved in the Oncogenic Properties of ZNF518B in Colorectal Cancer
    Epigenetic mechanisms are involved in the oncogenic properties of ZNF518B in colorectal cancer Francisco Gimeno-Valiente, Ángela L. Riffo-Campos, Luis Torres, Noelia Tarazona, Valentina Gambardella, Andrés Cervantes, Gerardo López-Rodas, Luis Franco and Josefa Castillo SUPPLEMENTARY METHODS 1. Selection of genomic sequences for ChIP analysis To select the sequences for ChIP analysis in the five putative target genes, namely, PADI3, ZDHHC2, RGS4, EFNA5 and KAT2B, the genomic region corresponding to the gene was downloaded from Ensembl. Then, zoom was applied to see in detail the promoter, enhancers and regulatory sequences. The details for HCT116 cells were then recovered and the target sequences for factor binding examined. Obviously, there are not data for ZNF518B, but special attention was paid to the target sequences of other zinc-finger containing factors. Finally, the regions that may putatively bind ZNF518B were selected and primers defining amplicons spanning such sequences were searched out. Supplementary Figure S3 gives the location of the amplicons used in each gene. 2. Obtaining the raw data and generating the BAM files for in silico analysis of the effects of EHMT2 and EZH2 silencing The data of siEZH2 (SRR6384524), siG9a (SRR6384526) and siNon-target (SRR6384521) in HCT116 cell line, were downloaded from SRA (Bioproject PRJNA422822, https://www.ncbi. nlm.nih.gov/bioproject/), using SRA-tolkit (https://ncbi.github.io/sra-tools/). All data correspond to RNAseq single end. doBasics = TRUE doAll = FALSE $ fastq-dump -I --split-files SRR6384524 Data quality was checked using the software fastqc (https://www.bioinformatics.babraham. ac.uk /projects/fastqc/). The first low quality removing nucleotides were removed using FASTX- Toolkit (http://hannonlab.cshl.edu/fastxtoolkit/).
    [Show full text]
  • Supplementary,Figure,1 Supplementary Figure 1
    A B A2058 (BRAF*) IMR90 3 +700% 5 +2400% % change vs. day 0 % change vs. day 0 ! DT$$$V$$$DTV ! DT$$E$$DTE 4 +1500% p!ERK 2 +300% p!ERK 3 +700% ERK ERK 2 +300% H3K9Ac 1 +100% H3K9Ac (day3 vs. day 0) (day 3 vs. day ) 1 +100% H3 H3 0 0 0 0 DMSO DT V DT+V DMSO DT ENT DT+ENT log2 fold change in cell number log2 fold change in cell number C D E Hs695THs695T Hs695T 2.040 2.0 Hs695T DMSODMSO NIH3T3 DMSO 293T 1.5 1.5 2.0 MEKi+BRAFi DTDT 5 +3100% 30 4 +1500% % change vs. day 0 DMSO % change vs. day 0 1.0 1.0 HDACi E E 4 +1500% 1.5 0.5 MEKi+BRAFi+HDACi DT 3 +700% 0.520 DTEDTE 3 +700% 0.0 1.0 E 20 40 60 0.080 2 +300% log2 fold growth -0.5 10 20 40 60 80 2 +300% hours of treatment 0.5 DTE log2 fold growth -0.5 -1.0 hours of treatment 1 +100% 1 +100% (day3 vs. day 0) (day3 vs. day 0) -1.00 0.0 0 0 0 20 2040 4060 8060 80 0 0 DMSO DT ENT DT+ENT Caspase 3/7 positive cells (%) DMSO DT ENT DT+ENT log2 fold growth -0.5hours of treatment log2 fold change in cell number hours of treatment log2 fold change in cell number -1.0 F G A2058'(high'MITF) SKMEL5'(high'MITF) Resistant Sensitive High'MITFMITF Expression 4.5 4.0 3.5 3.0 2.5 DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT 2.0 1.5 MITF MITF 1.0 0.5 Low'MITF Vinculin Vinculin 0.4 0.3 0.10 RPMI7951'(low'MITF) Hs695T'(low'MITF) 0.08 Resistant Sensitive 0.06 0.04 Relative MITF expression (MITF/Actin) 0.02 0.00 DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT DMSO MEKi VOR ENT MEKi+VOR MEKi+ENT A375 A2058 MEWO A101D Hs695T SKMEL5 SKMEL2 LOXIMVI MITF MITF UACC257 MALME3MRPMI7951 Vinculin Vinculin sensitive resistant H Hs695T ! E$$$$$$DTE HDAC1 HDAC2 HDAC3 GAPDH Supplementary,Figure,1 Supplementary Figure 1.
    [Show full text]
  • The Effect of Compound L19 on Human Colorectal Cells (DLD-1)
    Stephen F. Austin State University SFA ScholarWorks Electronic Theses and Dissertations Spring 5-16-2018 The Effect of Compound L19 on Human Colorectal Cells (DLD-1) Sepideh Mohammadhosseinpour [email protected] Follow this and additional works at: https://scholarworks.sfasu.edu/etds Part of the Biotechnology Commons Tell us how this article helped you. Repository Citation Mohammadhosseinpour, Sepideh, "The Effect of Compound L19 on Human Colorectal Cells (DLD-1)" (2018). Electronic Theses and Dissertations. 188. https://scholarworks.sfasu.edu/etds/188 This Thesis is brought to you for free and open access by SFA ScholarWorks. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of SFA ScholarWorks. For more information, please contact [email protected]. The Effect of Compound L19 on Human Colorectal Cells (DLD-1) Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License. This thesis is available at SFA ScholarWorks: https://scholarworks.sfasu.edu/etds/188 The Effect of Compound L19 on Human Colorectal Cells (DLD-1) By Sepideh Mohammadhosseinpour, Master of Science Presented to the Faculty of the Graduate School of Stephen F. Austin State University In Partial Fulfillment Of the Requirements For the Degree of Master of Science in Biotechnology STEPHEN F. AUSTIN STATE UNIVERSITY May, 2018 The Effect of Compound L19 on Human Colorectal Cells (DLD-1) By Sepideh Mohammadhosseinpour, Master of Science APPROVED: Dr. Beatrice A. Clack, Thesis Director Dr. Josephine Taylor, Committee Member Dr. Rebecca Parr, Committee Member Dr. Stephen Mullin, Committee Member Pauline Sampson, Ph.D.
    [Show full text]
  • Rad9 (RAD9A) Rabbit Polyclonal Antibody – TA308749 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA308749 Rad9 (RAD9A) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: IF, IHC, WB Recommended Dilution: ICC/IF:1:100-1:1000; IHC:1:100-1:1000; WB:1:500-1:3000 Reactivity: Human Host: Rabbit Isotype: IgG Clonality: Polyclonal Immunogen: Recombinant fragment contain a sequence corresponding to a region within amino acids 197 and 391 of Rad9 Formulation: 1XPBS, 1% BSA, 20% Glycerol (pH7). 0.01% Thimerosal was added as a preservative. Concentration: lot specific Purification: Purified by antigen-affinity chromatography. Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 43 kDa Gene Name: RAD9 checkpoint clamp component A Database Link: NP_004575 Entrez Gene 5883 Human Q99638 Background: This gene product is highly similar to Schizosaccharomyces pombe rad9, a cell cycle checkpoint protein required for cell cycle arrest and DNA damage repair in response to DNA damage. This protein is found to possess 3' to 5' exonuclease activity, which may contribute to its role in sensing and repairing DNA damage. It forms a checkpoint protein complex with RAD1 and HUS1. This complex is recruited by checkpoint protein RAD17 to the sites of DNA damage, which is thought to be important for triggering the checkpoint-signaling cascade. Use of alternative polyA sites has been noted for this gene. [provided by RefSeq] Synonyms: RAD9 This product is to be used for laboratory only.
    [Show full text]
  • Alterations of the Pro-Survival Bcl-2 Protein Interactome in Breast Cancer
    bioRxiv preprint doi: https://doi.org/10.1101/695379; this version posted July 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-NC-ND 4.0 International license. 1 Alterations of the pro-survival Bcl-2 protein interactome in 2 breast cancer at the transcriptional, mutational and 3 structural level 4 5 Simon Mathis Kønig1, Vendela Rissler1, Thilde Terkelsen1, Matteo Lambrughi1, Elena 6 Papaleo1,2 * 7 1Computational Biology Laboratory, Danish Cancer Society Research Center, 8 Strandboulevarden 49, 2100, Copenhagen 9 10 2Translational Disease Systems Biology, Faculty of Health and Medical Sciences, Novo 11 Nordisk Foundation Center for Protein Research University of Copenhagen, Copenhagen, 12 Denmark 13 14 Abstract 15 16 Apoptosis is an essential defensive mechanism against tumorigenesis. Proteins of the B-cell 17 lymphoma-2 (Bcl-2) family regulates programmed cell death by the mitochondrial apoptosis 18 pathway. In response to intracellular stresses, the apoptotic balance is governed by interactions 19 of three distinct subgroups of proteins; the activator/sensitizer BH3 (Bcl-2 homology 3)-only 20 proteins, the pro-survival, and the pro-apoptotic executioner proteins. Changes in expression 21 levels, stability, and functional impairment of pro-survival proteins can lead to an imbalance 22 in tissue homeostasis. Their overexpression or hyperactivation can result in oncogenic effects. 23 Pro-survival Bcl-2 family members carry out their function by binding the BH3 short linear 24 motif of pro-apoptotic proteins in a modular way, creating a complex network of protein- 25 protein interactions.
    [Show full text]