DOCSLIB.ORG

EFFECT of SODIUM BUTYRATE on HDAC8 Mrna EXPRESSION in COLORECTAL CANCER CELL LINES and MOLECULAR DOCKING STUDY of LHX1 - SODIUM BUTYRATE INTERACTION

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
EFFECT of SODIUM BUTYRATE on HDAC8 Mrna EXPRESSION in COLORECTAL CANCER CELL LINES and MOLECULAR DOCKING STUDY of LHX1 - SODIUM BUTYRATE INTERACTION EXCLI Journal 2020;19:1038-1051 – ISSN 1611-2156 Received: January 30, 2020, accepted: July 09, 2020, published: July 23, 2020 Original article: EFFECT OF SODIUM BUTYRATE ON HDAC8 mRNA EXPRESSION IN COLORECTAL CANCER CELL LINES AND MOLECULAR DOCKING STUDY OF LHX1 - SODIUM BUTYRATE INTERACTION Flora Forouzesh (PhD)a*, Mahsa Ghiaghi (MSc)b, Hamzeh Rahimi (PhD)c a Assistant professor, Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. E-mail: [email protected] b Student of Master Science of Genetics, Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. E-mail: [email protected] c Assistant professor, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran. E-mail: [email protected] * Corresponding author: Dr. Flora Forouzesh, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Dr Sharyati street, Shahid Khaghani street. P.O. Box: 193951495, Tehran, Iran. Tel: +98(21) 22006660-7, Fax: +98(21) 22600714, E-mail: [email protected], [email protected] http://dx.doi.org/10.17179/excli2020-2010 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/). ABSTRACT Colorectal cancer (CRC) is the third most common type of cancer and the fourth leading cause of cancer related deaths worldwide. The Histone Deacetylase 8 (HDAC8) gene is a gene with unique features which can be used as a potential target for drug design. The LHX1 transcription factor is an important transcription factor for this gene. The aim of this study was to investigate the effect of sodium butyrate (NaB) as a histone deacetylase inhib- itor (HDACi) on the expression of the HDAC8 gene in the colorectal cancer cell line, and the molecular docking of the LHX1 transcription factor with NaB. For this purpose, HCT-116 and HT-29 cell lines were treated with different concentrations of NaB (6.25 mM to 150 mM) at 24, 48 and 72 hours. Subsequently, RNA was extract- ed from the treated and untreated cells and cDNA was synthesized. Quantitative Real-Time-PCR was done to investigate the mRNA expression of HDAC8. Molecular docking was also performed to investigate the interac- tion between NaB and LHX1. Based on Real-time-PCR results, the concentration of 150 mM of NaB after 24 hours in HT-29 and HCT-116 cell lines caused a significant reduction in mRNA expression of HDAC8 (P<0.05). After 48 hours of treatment, there was a significant decrease in the mRNA expression of HDAC8 at all concentrations (P<0.05). The docking results showed that LHX1 and NaB interacted best at the lowest energy levels. Our results also showed that NaB bonded strongly to LHX1. In addition, our results demonstrated that NaB bound to the LHX1 transcription factor and inhibited the function of this factor and consequently decreased the transcription from the HDAC8 gene which resulted in cell death. Future studies are needed to assess the like- ly molecular mechanisms of NaB action on gene expression. Keywords: Colorectal cancer, sodium butyrate, HDAC8 gene, LHX1 transcription factor, docking, HT-29, HCT-116 cell lines 1038 EXCLI Journal 2020;19:1038-1051 – ISSN 1611-2156 Received: January 30, 2020, accepted: July 09, 2020, published: July 23, 2020 INTRODUCTION pressed in several types of tumors. The Colorectal cancer (CRC) is the fourth pharmacological inhibition of HDACs also most common cause of death from cancer in known as HDACi may be considered as new the world (Ansari et al., 2006; Mundade et method for cancer therapy (Bolden et al., al., 2014; Stigliano et al., 2014). As it has 2006; Minucci and Pelicci, 2006; Hildmann been found, cancer is the result of a wide et al., 2007; Riester et al., 2007; range of genetic and epigenetic changes. Shuttleworth et al., 2010). It has been ob- Histone acetylation is one of such epigenetic served that, HDACi can change the balance changes (Esteller, 2002; Johnstone, 2002; between HATs and HDACs and therefore Iizuka and Smith, 2003). Acetylation levels lead to a hyperacetylated state of histones are the result of the balance of the activities that causes cell cycle arrest, inducing tran- between histone deacetylase (HDAC) en- scriptional inhibition and apoptosis which are valid strategies for cancer treatment zymes and histone acetyltransferase (HAT) enzymes (Bannister and Kouzarides, 2011). (Bolden et al., 2006). The HDAC8 gene is a class I HDAC gene One of these HDACis is sodium butyrate (Hu et al., 2000; Van den Wyngaert et al., (NaB). Butyrate is produced by anaerobic 2000; Wawruszak et al., 2019), which is pre- bacterial fermentation of dietary fibers and is sent in the nucleus and cytoplasm of cells a short-chain fatty acid (Emenaker et al., distinctively (Waltregny et al., 2004; Li et 2001; Hinnebusch et al., 2002). One of the al., 2014). HDAC8 is expressed in normal functions of butyrate is the anti- tissues, but its expression increases in can- inflammatory effect that plays a role in in- cerous tissues (Song et al., 2015). For exam- hibiting histone deacetylase. Butyrate specif- ple, HDAC8 is correlated with T-cell lym- ically inhibits the class I HDAC. And it has phoma, childhood neuroblastoma and gas- the highest level of inhibition for HDAC8 tric, colon, lung, breast cancers. In addition, (Cummings and Macfarlane, 1991). Hence, HDAC8 shows functional abundance with the production of butyrate in the colon may HDAC6 when overexpressed in Hela and protect against colon cancer and inhibit the cervical cancer cells (Vanaja et al., 2018). As development of colon cancer (Emenaker et a result, HDAC8 can potentially be an attrac- al., 2001; He et al., 2001). As a result of such tive target for structure-based drug design findings, identifying the chemical properties because it exhibits a number of unique char- that govern the action of NaB on HDAC8 acteristics allowing the development of se- could be significant for the development of lective inhibitors. One particularly interest- new selective drugs with low side effects. It ing feature in this regard is the formation of is also known that logical drug design in- the foot pocket also called the acetate release volves investigating new molecules that complement the molecular target in struc- channel, which is a sub-pocket observed in class I HDACs, such as HDAC1, 2, 3 and 8, ture, aiming to reach an allosteric site or the but not in class II isotypes such as, HDAC4 active site (Bieliauskas and Pflum, 2008; Estiu et al., 2008, 2010; Nussinov and Tsai, and 7 X-ray structures (Hildmann et al., 2007). HDACs are regulators of gene ex- 2012). Moreover, molecular docking can be pression, that affect cell cycle arrest, apopto- defined as a simulation process where a lig- sis, angiogenesis, and the differentiation of and position is estimated at a predicted or different cell types (Hildmann et al., 2007; pre-defined binding site. This process can be Riester et al., 2007). Moreover, abnormal used widely; with minimal effort and ex- histone modification patterns have been pense, and it may increase the effectiveness found to be closely associated with several of obtaining potential drugs. Through mo- diseases such as cancers (Ma et al., 2019). lecular docking, we aim to clarify the benefi- According to the Human Protein Atlas cial HDAC8 structural features which may (2019), these types of class I HDACs are ex- help to obtain knowledge for drug develop- 1039 EXCLI Journal 2020;19:1038-1051 – ISSN 1611-2156 Received: January 30, 2020, accepted: July 09, 2020, published: July 23, 2020 ment in the future using some known selec- study between NaB and LHX1 as a tran- tive HDACis (Knegtel et al., 1997; Alonso et scription factor of HDAC8 was done in this al., 2006). study. The LHX1 protein is one of the transcrip- tion factors involved in the expression of the MATERIALS AND METHODS HDAC8 gene. The LIM homeobox 1 protein Cell culture of the human cell is encoded by the LHX1 HCT-116 (the human colorectal carci- gene. This protein is a transcription factor, noma) and HT-29 (the colorectal adenocar- important for control of the development and cinoma) cell lines were obtained from the differentiation of neural, lymphoid, renal and Iranian Biological Research Center (Pastor, urogenital cells. The LHX protein plays a Tehran, Iran). HCT-116 and HT-29 cell lines role in determining the origin and identity of were cultured in Dulbecco's modified Eagle's the cell. In humans, the encoding genome is medium (DMEM) and RPMI 1640, respec- located on chromosome 17q11.2-q12. Its tively, ( Gibco by life technologies, Germa- molecular weight is 44.808 Dalton and it ny) including 10 % fetal bovine serum (FBS; contains 406 amino acids. LHX1 binds to Gibco by life technologies, Germany) and DNA through its homeodomains and is an 1 % penicillin/streptomycin (Dacell, Iran) at important evolutionary regulator. The LHX1 37 °C and 5 % CO2. The cells were grown to protein contains 3 domains, which include: approximately 80 % confluence. A homeobox domain for DNA binding, and two LIM domains, which are involved in the Cell treatment and cytotoxicity assay ion-zinc connection. The LIM domains in- CRC cell lines were plated at 50×103 volved in zinc ion interconnection, play a cells per well in a 200 μL volume in 96-well role in cytoskeleton organization, cell adhe- plates. CRC cell lines were treated with the sion, epithelial development, gene transcrip- medium containing 6.25 to 200 mM of NaB, tion, mRNA trafficking, translation, protein (Biobasic, Canada Inc.) according to our folding, and chromatin remodeling. In addi- previous study, (Ghiaghi et al., 2019) for 24 tion, LIM domains have seven conserved and 48 hours.
Load more

Recommended publications
  • The Zinc-Finger Protein CNBP Is Required for Forebrain Formation In
    Development 130, 1367-1379 1367 © 2003 The Company of Biologists Ltd doi:10.1242/dev.00349 The zinc-finger protein CNBP is required for forebrain formation in the mouse Wei Chen1,2, Yuqiong Liang1, Wenjie Deng1, Ken Shimizu1, Amir M. Ashique1,2, En Li3 and Yi-Ping Li1,2,* 1Department of Cytokine Biology, The Forsyth Institute, Boston, MA 02115, USA 2Harvard-Forsyth Department of Oral Biology, Harvard School of Dental Medicine, Boston, MA 02115, USA 3Cardiovascular Research Center, Massachusetts General Hospital, Department of Medicine, Harvard Medical School, Charlestown, MA 02129, USA *Author for correspondence (e-mail: [email protected]) Accepted 19 December 2002 SUMMARY Mouse mutants have allowed us to gain significant insight (AME), headfolds and forebrain. In Cnbp–/– embryos, the into axis development. However, much remains to be visceral endoderm remains in the distal tip of the conceptus learned about the cellular and molecular basis of early and the ADE fails to form, whereas the node and notochord forebrain patterning. We describe a lethal mutation mouse form normally. A substantial reduction in cell proliferation strain generated using promoter-trap mutagenesis. The was observed in the anterior regions of Cnbp–/– embryos at mutants exhibit severe forebrain truncation in homozygous gastrulation and neural-fold stages. In these regions, Myc mouse embryos and various craniofacial defects in expression was absent, indicating CNBP targets Myc in heterozygotes. We show that the defects are caused by rostral head formation. Our findings demonstrate that disruption of the gene encoding cellular nucleic acid Cnbp is essential for the forebrain induction and binding protein (CNBP); Cnbp transgenic mice were able specification.
    [Show full text]
  • Identification of Candidate Genes and Pathways Associated with Obesity
    animals Article Identification of Candidate Genes and Pathways Associated with Obesity-Related Traits in Canines via Gene-Set Enrichment and Pathway-Based GWAS Analysis Sunirmal Sheet y, Srikanth Krishnamoorthy y , Jihye Cha, Soyoung Choi and Bong-Hwan Choi * Animal Genome & Bioinformatics, National Institute of Animal Science, RDA, Wanju 55365, Korea; [email protected] (S.S.); [email protected] (S.K.); [email protected] (J.C.); [email protected] (S.C.) * Correspondence: [email protected]; Tel.: +82-10-8143-5164 These authors contributed equally. y Received: 10 October 2020; Accepted: 6 November 2020; Published: 9 November 2020 Simple Summary: Obesity is a serious health issue and is increasing at an alarming rate in several dog breeds, but there is limited information on the genetic mechanism underlying it. Moreover, there have been very few reports on genetic markers associated with canine obesity. These studies were limited to the use of a single breed in the association study. In this study, we have performed a GWAS and supplemented it with gene-set enrichment and pathway-based analyses to identify causative loci and genes associated with canine obesity in 18 different dog breeds. From the GWAS, the significant markers associated with obesity-related traits including body weight (CACNA1B, C22orf39, U6, MYH14, PTPN2, SEH1L) and blood sugar (PRSS55, GRIK2), were identified. Furthermore, the gene-set enrichment and pathway-based analysis (GESA) highlighted five enriched pathways (Wnt signaling pathway, adherens junction, pathways in cancer, axon guidance, and insulin secretion) and seven GO terms (fat cell differentiation, calcium ion binding, cytoplasm, nucleus, phospholipid transport, central nervous system development, and cell surface) which were found to be shared among all the traits.
    [Show full text]
  • Comparative Transcriptomics Reveals Similarities and Differences
    Seifert et al. BMC Cancer (2015) 15:952 DOI 10.1186/s12885-015-1939-9 RESEARCH ARTICLE Open Access Comparative transcriptomics reveals similarities and differences between astrocytoma grades Michael Seifert1,2,5*, Martin Garbe1, Betty Friedrich1,3, Michel Mittelbronn4 and Barbara Klink5,6,7 Abstract Background: Astrocytomas are the most common primary brain tumors distinguished into four histological grades. Molecular analyses of individual astrocytoma grades have revealed detailed insights into genetic, transcriptomic and epigenetic alterations. This provides an excellent basis to identify similarities and differences between astrocytoma grades. Methods: We utilized public omics data of all four astrocytoma grades focusing on pilocytic astrocytomas (PA I), diffuse astrocytomas (AS II), anaplastic astrocytomas (AS III) and glioblastomas (GBM IV) to identify similarities and differences using well-established bioinformatics and systems biology approaches. We further validated the expression and localization of Ang2 involved in angiogenesis using immunohistochemistry. Results: Our analyses show similarities and differences between astrocytoma grades at the level of individual genes, signaling pathways and regulatory networks. We identified many differentially expressed genes that were either exclusively observed in a specific astrocytoma grade or commonly affected in specific subsets of astrocytoma grades in comparison to normal brain. Further, the number of differentially expressed genes generally increased with the astrocytoma grade with one major exception. The cytokine receptor pathway showed nearly the same number of differentially expressed genes in PA I and GBM IV and was further characterized by a significant overlap of commonly altered genes and an exclusive enrichment of overexpressed cancer genes in GBM IV. Additional analyses revealed a strong exclusive overexpression of CX3CL1 (fractalkine) and its receptor CX3CR1 in PA I possibly contributing to the absence of invasive growth.
    [Show full text]
  • The Role of Epigenomics in Osteoporosis and Osteoporotic Vertebral Fracture
    International Journal of Molecular Sciences Review The Role of Epigenomics in Osteoporosis and Osteoporotic Vertebral Fracture Kyoung-Tae Kim 1,2 , Young-Seok Lee 1,3 and Inbo Han 4,* 1 Department of Neurosurgery, School of Medicine, Kyungpook National University, Daegu 41944, Korea; [email protected] (K.-T.K.); [email protected] (Y.-S.L.) 2 Department of Neurosurgery, Kyungpook National University Hospital, Daegu 41944, Korea 3 Department of Neurosurgery, Kyungpook National University Chilgok Hospital, Daegu 41944, Korea 4 Department of Neurosurgery, CHA University School of medicine, CHA Bundang Medical Center, Seongnam-si, Gyeonggi-do 13496, Korea * Correspondence: [email protected]; Tel.: +82-31-780-1924; Fax: +82-31-780-5269 Received: 6 November 2020; Accepted: 8 December 2020; Published: 11 December 2020 Abstract: Osteoporosis is a complex multifactorial condition of the musculoskeletal system. Osteoporosis and osteoporotic vertebral fracture (OVF) are associated with high medical costs and can lead to poor quality of life. Genetic factors are important in determining bone mass and structure, as well as any predisposition for bone degradation and OVF. However, genetic factors are not enough to explain osteoporosis development and OVF occurrence. Epigenetics describes a mechanism for controlling gene expression and cellular processes without altering DNA sequences. The main mechanisms in epigenetics are DNA methylation, histone modifications, and non-coding RNAs (ncRNAs). Recently, alterations in epigenetic mechanisms and their activity have been associated with osteoporosis and OVF. Here, we review emerging evidence that epigenetics contributes to the machinery that can alter DNA structure, gene expression, and cellular differentiation during physiological and pathological bone remodeling.
    [Show full text]
  • Genetic Identification of Brain Cell Types Underlying Schizophrenia
    bioRxiv preprint doi: https://doi.org/10.1101/145466; this version posted June 2, 2017. 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. Genetic identification of brain cell types underlying schizophrenia Nathan G. Skene 1 †, Julien Bryois 2 †, Trygve E. Bakken3, Gerome Breen 4,5, James J Crowley 6, Héléna A Gaspar 4,5, Paola Giusti-Rodriguez 6, Rebecca D Hodge3, Jeremy A. Miller 3, Ana Muñoz-Manchado 1, Michael C O’Donovan 7, Michael J Owen 7, Antonio F Pardiñas 7, Jesper Ryge 8, James T R Walters 8, Sten Linnarsson 1, Ed S. Lein 3, Major Depressive Disorder Working Group of the Psychiatric Genomics Consortium, Patrick F Sullivan 2,6 *, Jens Hjerling- Leffler 1 * Affiliations: 1 Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden. 2 Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, SE-17177 Stockholm, Sweden. 3 Allen Institute for Brain Science, Seattle, Washington 98109, USA. 4 King’s College London, Institute of Psychiatry, Psychology and Neuroscience, MRC Social, Genetic and Developmental Psychiatry (SGDP) Centre, London, UK. 5 National Institute for Health Research Biomedical Research Centre, South London and Maudsley National Health Service Trust, London, UK. 6 Departments of Genetics, University of North Carolina, Chapel Hill, NC, 27599-7264, USA. 7 MRC Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical Neurosciences, School of Medicine, Cardiff University, Cardiff, UK.
    [Show full text]
  • Anti-LHX1 / LIM1 Antibody (ARG42944)
    Product datasheet [email protected] ARG42944 Package: 100 μl anti-LHX1 / LIM1 antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes LHX1 / LIM1 Tested Reactivity Hu, Ms, Rat Tested Application WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name LHX1 / LIM1 Antigen Species Human Immunogen Recombinant fusion protein corresponding to aa. 120-180 of Human LHX1 / LIM1 (NP_005559.2). Conjugation Un-conjugated Alternate Names LIM/homeobox protein Lhx1; Homeobox protein Lim-1; hLim-1; LIM1; LIM-1; LIM homeobox protein 1 Application Instructions Application table Application Dilution WB 1:500 - 1:2000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control Rat testis Calculated Mw 45 kDa Observed Size ~ 45 kDa Properties Form Liquid Purification Affinity purified. Buffer PBS (pH 7.3), 0.02% Sodium azide and 50% Glycerol. Preservative 0.02% Sodium azide Stabilizer 50% Glycerol Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. Note For laboratory research only, not for drug, diagnostic or other use. www.arigobio.com 1/2 Bioinformation Gene Symbol LHX1 Gene Full Name LIM homeobox 1 Background This gene encodes a member of a large protein family which contains the LIM domain, a unique cysteine-rich zinc-binding domain. The encoded protein is a transcription factor important for the development of the renal and urogenital systems.
    [Show full text]
  • The Lhx1-Ldb1 Complex Interacts with Furry to Regulate Microrna
    www.nature.com/scientificreports OPEN The Lhx1-Ldb1 complex interacts with Furry to regulate microRNA expression during pronephric Received: 2 July 2018 Accepted: 5 October 2018 kidney development Published: xx xx xxxx Eugenel B. Espiritu1, Amanda E. Crunk1, Abha Bais 1, Daniel Hochbaum2, Ailen S. Cervino3,4, Yu Leng Phua5, Michael B. Butterworth6, Toshiyasu Goto 7, Jacqueline Ho5, Neil A. Hukriede1,8 & M. Cecilia Cirio3,4 The molecular events driving specifcation of the kidney have been well characterized. However, how the initial kidney feld size is established, patterned, and proportioned is not well characterized. Lhx1 is a transcription factor expressed in pronephric progenitors and is required for specifcation of the kidney, but few Lhx1 interacting proteins or downstream targets have been identifed. By tandem- afnity purifcation, we isolated FRY like transcriptional coactivator (Fryl), one of two paralogous genes, fryl and furry (fry), have been described in vertebrates. Both proteins were found to interact with the Ldb1-Lhx1 complex, but our studies focused on Lhx1/Fry functional roles, as they are expressed in overlapping domains. We found that Xenopus embryos depleted of fry exhibit loss of pronephric mesoderm, phenocopying the Lhx1-depleted animals. In addition, we demonstrated a synergism between Fry and Lhx1, identifed candidate microRNAs regulated by the pair, and confrmed these microRNA clusters infuence specifcation of the kidney. Therefore, our data shows that a constitutively- active Ldb1-Lhx1 complex interacts with a broadly expressed microRNA repressor, Fry, to establish the kidney feld. In the vertebrate kidney, the specifcation of the renal progenitor cell feld is required for generating the appro- priate number of nephrons, as reduction of the number of renal progenitors results in reduced nephron endow- ment1.
    [Show full text]
  • LHX1 Gene LIM Homeobox 1
    LHX1 gene LIM homeobox 1 Normal Function The LHX1 gene provides instructions for making a protein that attaches (binds) to specific regions of DNA and regulates the activity of other genes. On the basis of this role, the protein produced from the LHX1 gene is called a transcription factor. The LHX1 protein is part of a large group of transcription factors called homeodomain proteins. The homeodomain is a region of the protein that allows it to bind to DNA. The LHX1 protein is found in many of the body's organs and tissues. Studies suggest that it plays particularly important roles in the development of the brain and female reproductive system. Health Conditions Related to Genetic Changes 17q12 deletion syndrome 17q12 deletion syndrome is a condition that results from the deletion of a small piece of chromosome 17 in each cell. Signs and symptoms of 17q12 deletion syndrome can include abnormalities of the kidneys, urinary tract, and reproductive system; a form of diabetes called maturity-onset diabetes of the young type 5 (MODY5); delayed development; intellectual disability; and behavioral or psychiatric disorders. Some females with this chromosomal change have Mayer-Rokitansky-Küster-Hauser syndrome, which is characterized by underdevelopment or absence of the vagina and uterus. Features associated with 17q12 deletion syndrome vary widely, even among affected members of the same family. The part of chromosome 17 that is deleted is on the long (q) arm of the chromosome at a position designated q12. This region of the chromosome contains 15 genes, including LHX1. A deletion of this region results in a loss of one copy of the LHX1 gene in each cell, leading to a reduced amount of LHX1 protein.
    [Show full text]
  • LHX1 (C-6): Sc-515631
    SANTA CRUZ BIOTECHNOLOGY, INC. LHX1 (C-6): sc-515631 BACKGROUND APPLICATIONS During development, genetically distinct subtypes of motor neurons express LHX1 (C-6) is recommended for detection of LHX1 of mouse, rat and human unique combinations of LIM-type homeodomain factors, which regulate cell origin by Western Blotting (starting dilution 1:100, dilution range 1:100- migration and guide motor axons to establish the fidelity of a binary choice 1:1000), immunoprecipitation [1-2 µg per 100-500 µg of total protein (1 ml in axonal trajectory. The LIM gene family encodes a set of gene products, of cell lysate)], immunofluorescence (starting dilution 1:50, dilution range which carry the LIM domain, a unique cysteine-rich zinc-binding domain. At 1:50-1:500) and solid phase ELISA (starting dilution 1:30, dilution range least 40 members of this family have been identified in vertebrates and 1:30-1:3000). invertebrates, and are distributed into 4 groups according to the number of Suitable for use as control antibody for LHX1 siRNA (h): sc-38708, LHX1 LIM domains and to the presence of homeodomains and kinase domains. The siRNA (m): sc-38709, LHX1 shRNA Plasmid (h): sc-38708-SH, LHX1 shRNA overlapping expression of LHX1, LHX3, LHX4, Isl-1 and Isl-2 in developing Plasmid (m): sc-38709-SH, LHX1 shRNA (h) Lentiviral Particles: sc-38708-V motorneurons along the spinal column may influence the establishment of and LHX1 shRNA (m) Lentiviral Particles: sc-38709-V. specific motorneuron subtypes. The human LHX1 gene maps to chromosome 17q12 and encodes a 384 amino acid protein.
    [Show full text]
  • Identification and Functional Analysis of a Novel LHX1 Mutation Associated with Congenital Absence of the Uterus and Vagina
    www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 5), pp: 8785-8790 Research Paper Identification and functional analysis of a novel LHX1 mutation associated with congenital absence of the uterus and vagina Wei Zhang1,2,3,*, Xueya Zhou4,5,*, Liyang Liu4, Ying Zhu1, Chunmei Liu6, Hong Pan3, Qiong Xing1, Jing Wang7, Xi Wang3, Xuegong Zhang4, Yunxia Cao1, Binbin Wang2,3 1Reproductive Medicine Center, The First Affiliated Hospital of Anhui Medical University, Hefei, China 2Graduate School, Peking Union Medical College, Beijing, China 3Center for Genetics, National Research Institute for Family Planning, Beijing, China 4MOE Key Laboratory of Bioinformatics, Bioinformatics Division and Center for Synthetic and Systems Biology, TNLIST/ Department of Automation, Tsinghua University, Beijing, China 5Department of Psychiatry and Centre for Genomic Sciences, Li KaShing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China 6Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Beijing, China 7Department of Medical Genetics, School of Basic Medical Sciences, Capital Medical University, Beijing, China *These authors have contributed equally to this work Correspondence to: Binbin Wang, email: [email protected] Yunxia Cao, email: [email protected] Keywords: LHX1, congenital absence of the uterus and vagina, Müllerian duct abnormality, whole exome sequencing, transcriptional activity Received: July 20, 2016 Accepted: October 22, 2016 Published: January 02, 2017 ABSTRACT Congenital absence of the uterus and vagina (CAUV) is the most extreme female Müllerian duct abnormality. Several researches proposed that genetic factors contributed to this disorder, whereas the precise genetic mechanism is far from full elucidation. Here, utilizing whole-exome sequencing (WES), we identified one novel missense mutation in LHX1 (NM_005568: c.G1108A, p.A370T) in one of ten unrelated patients diagnosed with CAUV.
    [Show full text]
  • LIM1 (LHX1) Mouse Monoclonal Antibody [Clone ID: OTI2G3] Product Data
    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 CF504534 LIM1 (LHX1) Mouse Monoclonal Antibody [Clone ID: OTI2G3] Product data: Product Type: Primary Antibodies Clone Name: OTI2G3 Applications: FC, IF, WB Recommended Dilution: WB 1:2000, IF 1:100, FLOW 1:100 Reactivity: Human, Mouse, Rat Host: Mouse Isotype: IgG1 Clonality: Monoclonal Immunogen: Human recombinant protein fragment corresponding to amino acids 100-362 of human LHX1(NP_005559) produced in E.coli. Formulation: Lyophilized powder (original buffer 1X PBS, pH 7.3, 8% trehalose) Reconstitution Method: For reconstitution, we recommend adding 100uL distilled water to a final antibody concentration of about 1 mg/mL. To use this carrier-free antibody for conjugation experiment, we strongly recommend performing another round of desalting process. (OriGene recommends Zeba Spin Desalting Columns, 7KMWCO from Thermo Scientific) Purification: Purified from mouse ascites fluids or tissue culture supernatant by affinity chromatography (protein A/G) Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 44.6 kDa Gene Name: Homo sapiens LIM homeobox 1 (LHX1), mRNA. Database Link: NP_005559 Entrez Gene 16869 MouseEntrez Gene 257634 RatEntrez Gene 3975 Human P48742 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 3 LIM1 (LHX1) Mouse Monoclonal Antibody [Clone ID: OTI2G3] – CF504534 Background: This gene encodes a member of a large protein family which contains the LIM domain, a unique cysteine-rich zinc-binding domain.
    [Show full text]
  • The Spalt Family Transcription Factor Sall3 Regulates the Development Of
    RESEARCH ARTICLE 2325 Development 138, 2325-2336 (2011) doi:10.1242/dev.061846 © 2011. Published by The Company of Biologists Ltd The Spalt family transcription factor Sall3 regulates the development of cone photoreceptors and retinal horizontal interneurons Jimmy de Melo1, Guang-Hua Peng2, Shiming Chen2,3 and Seth Blackshaw1,4,5,6,7,* SUMMARY The mammalian retina is a tractable model system for analyzing transcriptional networks that guide neural development. Spalt family zinc-finger transcription factors play a crucial role in photoreceptor specification in Drosophila, but their role in mammalian retinal development has not been investigated. In this study, we show that that the spalt homolog Sall3 is prominently expressed in developing cone photoreceptors and horizontal interneurons of the mouse retina and in a subset of cone bipolar cells. We find that Sall3 is both necessary and sufficient to activate the expression of multiple cone-specific genes, and that Sall3 protein is selectively bound to the promoter regions of these genes. Notably, Sall3 shows more prominent expression in short wavelength-sensitive cones than in medium wavelength-sensitive cones, and that Sall3 selectively activates expression of the short but not the medium wavelength-sensitive cone opsin gene. We further observe that Sall3 regulates the differentiation of horizontal interneurons, which form direct synaptic contacts with cone photoreceptors. Loss of function of Sall3 eliminates expression of the horizontal cell-specific transcription factor Lhx1, resulting in a radial displacement of horizontal cells that partially phenocopies the loss of function of Lhx1. These findings not only demonstrate that Spalt family transcription factors play a conserved role in regulating photoreceptor development in insects and mammals, but also identify Sall3 as a factor that regulates terminal differentiation of both cone photoreceptors and their postsynaptic partners.
    [Show full text]