DOCSLIB.ORG

Supporting Information

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Supporting Information Supporting Information Jin et al. 10.1073/pnas.1418629112 SI Materials and Methods Santa Cruz Biotechnology, 1:100), anti–neural-specific β-tubulin − − − − Animals. Mice carrying the Gas1 (1), Shh (2), Cdo , Boc (3), (Tuj1, mouse, Millipore, 1:800), anti-Neurotrophin receptor P75 f − Smo (4), Gnaz (5), and transgenic Wnt1:Cre (6) alleles were (Rabbit, Millipore, 1:200), anti-GFP (rabbit, Invitrogen, 1:100), − − − previously described. For simplicity, Gas1 , Cdo ,andBoc were anti-mouse Gas1 (goat, R&D Systems, 1:200), and anti-HuC/D used for the Lac-Z knock-in allele of Gas1 and β-geo–internal (mouse, Molecular Probes, 1:100). Alexa Fluor 488- and Alexa ribosomal entry site (IRES) human placental alkaline phosphatase Fluor 568-conjugated secondary antibodies (Invitrogen) against (hPLAP) gene-trapped alleles of Cdo and Boc, respectively. Ap- specific species (goat, mouse, and rabbit) were used for detection propriate mating schemes were designed to generate embryos of (Molecular Probes, all at 1:1,000). DAPI (Sigma) was used at desired genotypes, including controls. Embryo stages are specified 1 μg/mL for staining of DNA. in the text. The vaginal plug date is designated as embryonic day 0.5 (E0.5), following convention. For genotyping, tail or embryo Neurosphere Culture. Neurosphere-like bodies were generated −/− sac DNAs were used. Oligonucleotide primers and conditions using whole guts dissected from E11.5 wild-type or Gas1 for PCR are described in corresponding publications and on the embryos as previously described (7–9). They were dissociated in f Jackson Laboratory (JAX) website. The Gas1 allele was gener- basal media by mechanical pipetting, then plated into culture ated for this work, and its characterization is detailed in Fig. S2; dish freshly coated with 20 μg/mL of fibronectin (Sigma), and ′ primers flanking the 3 loxP site were used for routine genotyping: cultured at 37 °C, 5% CO2. The fresh basal media was replaced 5′-TTGCCCCACGGTCCCGGGCGCA and 5′-CATGTTGG- every 2 d. Around day 5, 20 ng/mL of human recombinant epi- CTGCGGTACGAGCTG. All procedures are approved by the dermal growth factor (Calbiochem) was added to induce enteric Carnegie Institutional Animal Care and Use Committee. neurosphere formation. These primary neurospheres were then used for axon outgrowth and guidance assays. Histochemistry and Immunostaining. For X-gal reactions and im- munofluorescence, embryos or dissected guts were fixed in 4% Outgrowth and Turning Assays. For axon outgrowth assay, neuro- (wt/vol) paraformaldyhyde/PBS overnight at 4 °C, washed exten- spheres obtained from E11.5 guts described above were embedded sively in PBS, transferred through serial sucrose/PBS, and em- in the 3D collagen matrices (BD Biosciences) with a supplement bedded in O.C.T. compound (Tissue Tek) for cryosectioning. of different concentrations of recombinant Shh-N or GDNF Tissue sections 10 μm thick were made by using LEICA CM3050S proteins (specified in the text and figure legends) in basal media cryostat. X-gal reactions followed standard protocols as described for 48 h. For the turning assay, COS cell aggregates transfected for 24 h of staining and were counterstained with Nuclear Fast with a control or Shh expression plasmid by X-tremeGENE HP Red (Sigma), dehydrated, and mounted in Permount (Fisher) (7). (Roche) or heparin beads (Sigma) soaked in PBS or in PBS with For gut cross-section immunostaining, the sections were per- recombinant Shh-N (2 μg/mL, R&D Systems) were embedded in meablized in 1% Triton-X100 (Sigma)/PBS for 15 min at room 3D collagen matrices (10) next to neurospheres and cultured for temperature, blocked for 1 h in 4% (wt/vol) BSA (Sigma)/PBS 48 h. PTX (Calbiochem) at 75 ng/mL was added in the culture solution at room temperature, and then incubated with primary media. At the end of culture, samples were fixed and stained as antibodies in the blocking solution overnight at 4 °C. The next day, described above. Based on Tuj1 staining of the axon, axon length slides were washed three times with PBS (10 min per wash), was measured with MetaMorph software, and axon turning angle incubated with secondary antibodies for 1.5 h at room tempera- degrees were measured with ImageJ software. ture, washed with PBS three times, counterstained by DAPI for 5 min, and washed with PBS three times before mounting in Flu- RNA-Seq and qRT-PCR. Neurospheres in an undifferentiated or oromount G (SouthernBiotech). For single neuron immunostain- differentiated (induced by 50 ng/mL of GDNF for 48 h) status ing, dissociated neurons from E11.5 gut were cultured in eight-well were collected for RNA-seq. Total RNA was extracted using the chamber slides (Nunc) for 48 h in basal media plus 50 ng/mL of ArcturusPicoPure RNA isolation kit (Applied Biosystems). Then GDNF (Prospec). Basal media includes DMEM media (Life ribosomal RNA was depleted using the Ribo-Zero rRNA Re- Technology) supplied with 15% chicken embryo extract, 1% moval Core Kit (Epicentre). cDNA libraries were constructed pencillin/streptomycin (Life Technology), 0.1% glutamine (Life from ribonsome-depleted RNA using Illumina TruSeq RNA Technology), 1% N2 supplement (Life Technology), 2% B27 sup- Library Prep Kit v2 and sequenced using a HiSeq2000. RNA-seq plement (Life Technology), 50 μM β-mercaptoethanol (Gibco), 20 ng/mL bFGF (R&D Systems), and 35 ng/mL retinoic acid reads were mapped using TopHat to the mouse genome (mm9), (Sigma) per refs. 7–9. At the end of culture, samples were fixed and RefSeq annotations were downloaded from the University of in 4% paraformaldyhyde/PBS for 30 min at room temperature, California Santa Cruz table browser. The number of reads over- washed in PBS three times (10 min per wash), and the rest lapping exon regions of each gene was counted with custom scripts. Gαi followed the immunostaining protocol mentioned above. For Differentially expressed family members were analyzed using Gnaz neurosphere whole-mount immunostaining, cultured neurosphere edgeR. qRT-PCR was applied to confirm the gene expres- samples were fixed in 4% paraformaldyhyde/PBS overnight at sion. For qRT-PCR, 500 pg of total RNA from each sample was 4 °C, washed three times in PBS (20 min/wash) at room tem- used for standard RT using quantiTect reverse transcription kit perature, blocked for 2 h in 4% BSA solution at room temper- (Qiagen) in a 20-μL reaction. We then used 1 μLoftheRTre- ature, incubated with primary antibody overnight at 4 °C, washed action in a 20-μL PCR containing syber green for 45 cycles in three times in PBS (2 h per wash) at room temperature, in- C1000 Touch C (BioRad). The oligonucleotide primers used for cubated with secondary antibody overnight at 4 °C, washed three each gene are as follows: Gli1,5′-CCAAGCCAACTTTATGT- times in PBS (2 h per wash) at room temperature, and then CAGGG and 5′-AGCCCGCTTCTTTGTTAATTTGA; Gnaz,5′- imaged using a Nikon Eclipse TE200 fluorescence scope. CCCAGCAGGAGAAGTTTGATTTC and 5′-ATGTCGGCAA- The primary antibodies used were anti–β-gal (rabbit, Chem- AGCTCAGAGG; and Gapdh,5′-AGGTCGGTGTGAACGGA- icon, 1:1,000, or mouse, Promega, 1:1,000), anti-Gnaz (rabbit, TTTG and 5′-TGTAGACCATGTAGTTGAGGTCA. Relative Jin et al. www.pnas.org/cgi/content/short/1418629112 1of6 expression levels or Δcq values (Gli1 cq value – Gapdh cq) were buffer) per lane for SDS/PAGE. PVDF membranes (Bio-Rad) shown and are described in the figure legends. were used for Western blotting. Primary antibodies used were goat anti–Shh-N (N-19, Santa Cruz Biotechnology, 1:1,000), goat anti- Lentiviral shRNA Transduction. All lentiviral shRNA (of the GIPZ mGas1 (R&D Systems, 1:500), and mouse anti-Gapdh (clone 6C5, and pLEX system)-expressing vectors were purchased from Millipore, 1:5,000), followed by species-specific HRP-conjugated Thermo Scientific. Trans-Lentiviral shRNA Packaging Kit secondary antibodies and ECL detection (Amersham). The pro- (TL5913) was used to produce lentiviruses expressing mouse cedure was adapted from Biau et al. (7). Gnaz shRNAs (1, V2LMM-78357; 2, V2LMM-80266) and con- + trol nonspecific shRNA (nonshRNA, RHS4346) according to the Quantitation and Statistical Analyses. Villi containing Tuj1 - procedures provided by the company. The pLEX vector (Thermo stained axons were counted on captured digital images from Scientific) was used to express Smo-GFP and the dominant 10 sections of each of three embryos of each genotype. For in negative form of Gnaz (11) using the corresponding viral pack- vitro neurosphere assay, each experiment was repeated at aging kit (Thermo Scientific). Neurospheres were cultured in basal media with these lentiviral particles for 48 h and then used least three times, with 20 neurospheres prepared from at least for axon guidance assays with PBS- and Shh-N–soaked heparin three animals for each experiment. Bar graphs represent ± beads as described above. mean SEs. All statistical data considered significant were with P values <0.05, <0.01, or <0.001 as assessed by Student’s t Western Blots. Supernatants collected from transfected COS cells test; ns, not significant. They are presented in the figure leg- (48 h after transfection) were used neat at 7.5 μL (in SDS sample ends accordingly. 1. Martinelli DC, Fan CM (2007) Gas1 extends the range of Hedgehog action by facili- 7. Biau S, Jin S, Fan CM (2013) Gastrointestinal defects of the Gas1 mutant involve tating its signaling. Genes Dev 21(10):1231–1243. dysregulated Hedgehog and Ret signaling. Biol Open 2(2):144–155. 2. Chiang C, et al. (1996) Cyclopia and defective axial patterning in mice lacking Sonic 8. Fu M, Lui VC, Sham MH, Pachnis V, Tam PK (2004) Sonic hedgehog regulates the hedgehog gene function. Nature 383(6599):407–413. proliferation, differentiation, and migration of enteric neural crest cells in gut.
Load more

Recommended publications
  • Germline Mutations Affecting Gα11 in Hypoparathyroidism
    T h e new england journal o f medicine of a meta-analysis of studies of the effect of re- terone levels were increased. Although long- duced dietary salt on the incidence of cardiovas- term, modest reductions in salt intake result in cular events and death.1 The authors of the Co- small, physiologic increases in plasma renin ac- chrane report wrote that there was “no strong tivity,3 the preponderance of evidence suggests evidence of benefit.” In a summary statement, we that a reduced salt intake is associated with a wrote that this particular Cochrane analysis con- decreased risk of cardiovascular events and cluded that reducing dietary salt intake did not death. Furthermore, it is worth remembering decrease the risk of death or cardiovascular dis- that diuretics remain one of the most effective ease. Stigler et al. suggest that “indeterminate antihypertensive therapies, and their beneficial results,” rather than no significant effect, would effect on cardiovascular disease is well docu- be a more appropriate interpretation of the analy- mented.4 Nevertheless, as we suggested, in sis. Both interpretations may be correct. Although terms of safety, the lower limit of salt consump- it may not be possible to reject the null hypothe- tion has not been clearly defined. sis with certainty (i.e., no effect of reduced salt), Theodore A. Kotchen, M.D. the analysis should have been powered to detect Allen W. Cowley, Jr., Ph.D. a clinically meaningful difference, and the con- Medical College of Wisconsin clusion of no effect provides more guidance than Milwaukee, WI “indeterminate results” to clinical decision mak- [email protected] ers.
    [Show full text]
  • Supplemental Material
    Supplemental Table B ARGs in alphabetical order Symbol Title 3 months 6 months 9 months 12 months 23 months ANOVA Direction Category 38597 septin 2 1557 ± 44 1555 ± 44 1579 ± 56 1655 ± 26 1691 ± 31 0.05219 up Intermediate 0610031j06rik kidney predominant protein NCU-G1 491 ± 6 504 ± 14 503 ± 11 527 ± 13 534 ± 12 0.04747 up Early Adult 1G5 vesicle-associated calmodulin-binding protein 662 ± 23 675 ± 17 629 ± 16 617 ± 20 583 ± 26 0.03129 down Intermediate A2m alpha-2-macroglobulin 262 ± 7 272 ± 8 244 ± 6 290 ± 7 353 ± 16 0.00000 up Midlife Aadat aminoadipate aminotransferase (synonym Kat2) 180 ± 5 201 ± 12 223 ± 7 244 ± 14 275 ± 7 0.00000 up Early Adult Abca2 ATP-binding cassette, sub-family A (ABC1), member 2 958 ± 28 1052 ± 58 1086 ± 36 1071 ± 44 1141 ± 41 0.05371 up Early Adult Abcb1a ATP-binding cassette, sub-family B (MDR/TAP), member 1A 136 ± 8 147 ± 6 147 ± 13 155 ± 9 185 ± 13 0.01272 up Midlife Acadl acetyl-Coenzyme A dehydrogenase, long-chain 423 ± 7 456 ± 11 478 ± 14 486 ± 13 512 ± 11 0.00003 up Early Adult Acadvl acyl-Coenzyme A dehydrogenase, very long chain 426 ± 14 414 ± 10 404 ± 13 411 ± 15 461 ± 10 0.01017 up Late Accn1 amiloride-sensitive cation channel 1, neuronal (degenerin) 242 ± 10 250 ± 9 237 ± 11 247 ± 14 212 ± 8 0.04972 down Late Actb actin, beta 12965 ± 310 13382 ± 170 13145 ± 273 13739 ± 303 14187 ± 269 0.01195 up Midlife Acvrinp1 activin receptor interacting protein 1 304 ± 18 285 ± 21 274 ± 13 297 ± 21 341 ± 14 0.03610 up Late Adk adenosine kinase 1828 ± 43 1920 ± 38 1922 ± 22 2048 ± 30 1949 ± 44 0.00797 up Early
    [Show full text]
  • 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]
  • Ubiquitylome Profiling of Parkin-Null Brain Reveals Dysregulation Of
    Neurobiology of Disease 127 (2019) 114–130 Contents lists available at ScienceDirect Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi Ubiquitylome profiling of Parkin-null brain reveals dysregulation of calcium T homeostasis factors ATP1A2, Hippocalcin and GNA11, reflected by altered firing of noradrenergic neurons Key J.a,1, Mueller A.K.b,1, Gispert S.a, Matschke L.b, Wittig I.c, Corti O.d,e,f,g, Münch C.h, ⁎ ⁎ Decher N.b, , Auburger G.a, a Exp. Neurology, Goethe University Medical School, 60590 Frankfurt am Main, Germany b Institute for Physiology and Pathophysiology, Vegetative Physiology and Marburg Center for Mind, Brain and Behavior - MCMBB; Clinic for Neurology, Philipps-University Marburg, 35037 Marburg, Germany c Functional Proteomics, SFB 815 Core Unit, Goethe University Medical School, 60590 Frankfurt am Main, Germany d Institut du Cerveau et de la Moelle épinière, ICM, Paris, F-75013, France e Inserm, U1127, Paris, F-75013, France f CNRS, UMR 7225, Paris, F-75013, France g Sorbonne Universités, Paris, F-75013, France h Institute of Biochemistry II, Goethe University Medical School, 60590 Frankfurt am Main, Germany ARTICLE INFO ABSTRACT Keywords: Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among Parkinson's disease its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin Mitochondria triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear howse- Parkin lective neural circuits become vulnerable and finally undergo atrophy. Ubiquitin We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein Calcium targets of Parkin activity, an ubiquitin E3 ligase.
    [Show full text]
  • Predicting Coupling Probabilities of G-Protein Coupled Receptors Gurdeep Singh1,2,†, Asuka Inoue3,*,†, J
    Published online 30 May 2019 Nucleic Acids Research, 2019, Vol. 47, Web Server issue W395–W401 doi: 10.1093/nar/gkz392 PRECOG: PREdicting COupling probabilities of G-protein coupled receptors Gurdeep Singh1,2,†, Asuka Inoue3,*,†, J. Silvio Gutkind4, Robert B. Russell1,2,* and Francesco Raimondi1,2,* 1CellNetworks, Bioquant, Heidelberg University, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany, 2Biochemie Zentrum Heidelberg (BZH), Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, 3Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan and 4Department of Pharmacology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA Received February 10, 2019; Revised April 13, 2019; Editorial Decision April 24, 2019; Accepted May 01, 2019 ABSTRACT great use in tinkering with signalling pathways in living sys- tems (5). G-protein coupled receptors (GPCRs) control multi- Ligand binding to GPCRs induces conformational ple physiological states by transducing a multitude changes that lead to binding and activation of G-proteins of extracellular stimuli into the cell via coupling to situated on the inner cell membrane. Most of mammalian intra-cellular heterotrimeric G-proteins. Deciphering GPCRs couple with more than one G-protein giving each which G-proteins couple to each of the hundreds receptor a distinct coupling profile (6) and thus specific of GPCRs present in a typical eukaryotic organism downstream cellular responses. Determining these coupling is therefore critical to understand signalling. Here, profiles is critical to understand GPCR biology and phar- we present PRECOG (precog.russelllab.org): a web- macology. Despite decades of research and hundreds of ob- server for predicting GPCR coupling, which allows served interactions, coupling information is still missing for users to: (i) predict coupling probabilities for GPCRs many receptors and sequence determinants of coupling- specificity are still largely unknown.
    [Show full text]
  • Quantification of Cardiovascular Disease Biomarkers in Human
    proteomes Article Quantification of Cardiovascular Disease Biomarkers in Human Platelets by Targeted Mass Spectrometry Sebastian Malchow ID , Christina Loosse, Albert Sickmann and Christin Lorenz * Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., 44139 Dortmund, Germany; [email protected] (S.M.); [email protected] (C.L.); [email protected] (A.S.) * Correspondence: [email protected]; Tel.: +49-231-1392-289 Received: 3 October 2017; Accepted: 13 November 2017; Published: 15 November 2017 Abstract: Platelets are known to be key players in thrombosis and hemostasis, contributing to the genesis and progression of cardiovascular diseases. Due to their pivotal role in human physiology and pathology, platelet function is regulated tightly by numerous factors which have either stimulatory or inhibitory effects. A variety of factors, e.g., collagen, fibrinogen, ADP, vWF, thrombin, and thromboxane promote platelet adhesion and aggregation by utilizing multiple intracellular signal cascades. To quantify platelet proteins for this work, a targeted proteomics workflow was applied. In detail, platelets are isolated and lyzed, followed by a tryptic protein digest. Subsequently, a mix of stable isotope-labeled peptides of interesting biomarker proteins in concentrations ranging from 0.1 to 100 fmol is added to 3 µg digest. These peptides are used as an internal calibration curve to accurately quantify endogenous peptides and corresponding proteins in a pooled platelet reference sample by nanoLC-MS/MS with parallel reaction monitoring. In order to assure a valid quantification, limit of detection (LOD) and limit of quantification (LOQ), as well as linear range, were determined. This quantification of platelet activation and proteins by targeted mass spectrometry may enable novel diagnostic strategies in the detection and prevention of cardiovascular diseases.
    [Show full text]
  • Genomic Instability and DNA Ploidy Are Linked to DNA Copy Number Aberrations of 8P23 and 22Q11.23 in Gastric Cancers
    333-339.qxd 15/7/2010 11:08 Ì ™ÂÏ›‰·333 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 26: 333-339, 2010 333 Genomic instability and DNA ploidy are linked to DNA copy number aberrations of 8p23 and 22q11.23 in gastric cancers SHIGETO KAWAUCHI1, TOMOKO FURUAY1, TETSUJI UCHIYAMA2, ATSUSHI ADACHI2, TAKAE OKADA1 MOTONAO NAKAO1, ATSUNORI OGA1, KENICHIRO UCHIDA1 and KOHSUKE SASAKI1 1Department of Pathology, Yamaguchi University Graduate School of Medicine, Ube 755-8505; 2Department of Surgery, Iwakuni Medical Center, Iwakuni 740-0021, Japan Received April 8, 2010; Accepted June 2, 2010 DOI: 10.3892/ijmm_00000470 Abstract. The close relationship between chromosomal Introduction instability (CIN) and aneuploidy has been reported. The purpose of this study was to identify genomic aberrations Cancer progression is accompanied by the accumulation of present with CIN and aneuploidy in gastric cancers. FISH genetic alterations in the genes controlling cell proliferation and image cytometry were applied to 27 sporadic gastric and death. Indeed, some genomic abnormalities, ranging adenocarcinomas to identify CIN-positive tumors and to from point mutations to chromosomal aberrations, are determine DNA ploidy, respectively. In addition, array-based detected in virtually all cancers, including gastric cancers. comparative genomic hybridization was used to identify Although the biological characteristics of cancers greatly bacterial artificial chromosome clones that displayed vary from case to case, they are primarily affected by genomic differences in the frequency of copy number aberrations alterations. In general, cancer cells inherently take on genomic between CIN-positive and CIN-negative tumors, and instability that is conceptually divided into microsatellite between aneuploid and diploid tumors. There were many instability (MIN) and chromosomal instability (CIN) (1,2).
    [Show full text]
  • Cilia Gene Expression Patterns in Cancer MAX SHPAK 1,2 , MARCUS M
    CANCER GENOMICS & PROTEOMICS 11 : 13-24 (2014) Cilia Gene Expression Patterns in Cancer MAX SHPAK 1,2 , MARCUS M. GOLDBERG 2 and MATTHEW C. COWPERTHWAITE 3 1NeuroTexas Institute, St. David’s Health Care, Austin, TX, U.S.A.; 2Center for Systems and Synthetic Biology, University of Texas, Austin, TX, U.S.A.; 3Texas Advanced Computing Center, Austin, TX, U.S.A. Abstra ct. Non-motile cilia are thought to be important Cilia are organelles present on the surface of the majority of determinants of the progression of many types of cancers. human cell types. There are two classes of cilia: motile and Our goal was to identify patterns of cilia gene dysregulation non-motile. Non-motile (primary) cilia are generally sensory in eight cancer types (glioblastoma multiforme, colon organelles that are involved in signal transduction, response adenocarcinoma, breast adenocarcinoma, kidney renal clear to chemicals in the external environment, and cellular growth cell carcinoma, lung squamous cell carcinoma, lung and differentiation (1). Cilia also play an important adenocarcinoma, rectal adenocarcinoma, and ovarian developmental role in tissue and organ patterning, including cancer) profiled by The Cancer Genome Atlas. Among these cell adhesion/communication in the brain and the heart. types, 2.5-19.8% of cilia-associated genes were significantly Unsurprisingly, primary cilia are associated with a number differentially expressed (versus 5.5-32.4% dysregulation of human diseases, or ciliopathies, such as polycystic kidney across all genes). In four canc er types (breast disease, Bardet–Biedel syndrome, and Meckel–Gruber adenocarcinoma, colon adenocarcinoma, glioblastoma syndrome (2). multiforme, and ovarian cancer), cilia genes were on Primary cilia have also been implicated in cancer, and average down-reg ulated (median fold change from –1.53- tumorigenesis in particular, as a consequence of the –0.3), in the other four types, cilia genes were up- involvement of primary cilia in cell-cycle regulation.
    [Show full text]
  • G-Protein ␤␥-Complex Is Crucial for Efficient Signal Amplification in Vision
    The Journal of Neuroscience, June 1, 2011 • 31(22):8067–8077 • 8067 Cellular/Molecular G-Protein ␤␥-Complex Is Crucial for Efficient Signal Amplification in Vision Alexander V. Kolesnikov,1 Loryn Rikimaru,2 Anne K. Hennig,1 Peter D. Lukasiewicz,1 Steven J. Fliesler,4,5,6,7 Victor I. Govardovskii,8 Vladimir J. Kefalov,1 and Oleg G. Kisselev2,3 1Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, Departments of 2Ophthalmology and 3Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104, 4Research Service, Veterans Administration Western New York Healthcare System, and Departments of 5Ophthalmology (Ross Eye Institute) and 6Biochemistry, University at Buffalo/The State University of New York (SUNY), and 7SUNY Eye Institute, Buffalo, New York 14215, and 8Sechenov Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Saint Petersburg 194223, Russia A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the ␣-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary ␤␥-complex in this process remains a mystery. Here, we show that elimination of the transducin ␥-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin ␤␥-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.
    [Show full text]
  • Recurrent GNAQ Mutation Encoding T96S in Natural Killer/T Cell Lymphoma
    ARTICLE https://doi.org/10.1038/s41467-019-12032-9 OPEN Recurrent GNAQ mutation encoding T96S in natural killer/T cell lymphoma Zhaoming Li 1,2,9, Xudong Zhang1,2,9, Weili Xue1,3,9, Yanjie Zhang1,3,9, Chaoping Li1,3, Yue Song1,3, Mei Mei1,3, Lisha Lu1,3, Yingjun Wang1,3, Zhiyuan Zhou1,3, Mengyuan Jin1,3, Yangyang Bian4, Lei Zhang1,2, Xinhua Wang1,2, Ling Li1,2, Xin Li1,2, Xiaorui Fu1,2, Zhenchang Sun1,2, Jingjing Wu1,2, Feifei Nan1,2, Yu Chang1,2, Jiaqin Yan1,2, Hui Yu1,2, Xiaoyan Feng1,2, Guannan Wang5, Dandan Zhang5, Xuefei Fu6, Yuan Zhang7, Ken H. Young8, Wencai Li5 & Mingzhi Zhang1,2 1234567890():,; Natural killer/T cell lymphoma (NKTCL) is a rare and aggressive malignancy with a higher prevalence in Asia and South America. However, the molecular genetic mechanisms underlying NKTCL remain unclear. Here, we identify somatic mutations of GNAQ (encoding the T96S alteration of Gαq protein) in 8.7% (11/127) of NKTCL patients, through whole- exome/targeted deep sequencing. Using conditional knockout mice (Ncr1-Cre-Gnaqfl/fl), we demonstrate that Gαqdeficiency leads to enhanced NK cell survival. We also find that Gαq suppresses tumor growth of NKTCL via inhibition of the AKT and MAPK signaling pathways. Moreover, the Gαq T96S mutant may act in a dominant negative manner to promote tumor growth in NKTCL. Clinically, patients with GNAQ T96S mutations have inferior survival. Taken together, we identify recurrent somatic GNAQ T96S mutations that may contribute to the pathogenesis of NKTCL. Our work thus has implications for refining our understanding of the genetic mechanisms of NKTCL and for the development of therapies.
    [Show full text]
  • 140503 IPF Signatures Supplement Withfigs Thorax
    Supplementary material for Heterogeneous gene expression signatures correspond to distinct lung pathologies and biomarkers of disease severity in idiopathic pulmonary fibrosis Daryle J. DePianto1*, Sanjay Chandriani1⌘*, Alexander R. Abbas1, Guiquan Jia1, Elsa N. N’Diaye1, Patrick Caplazi1, Steven E. Kauder1, Sabyasachi Biswas1, Satyajit K. Karnik1#, Connie Ha1, Zora Modrusan1, Michael A. Matthay2, Jasleen Kukreja3, Harold R. Collard2, Jackson G. Egen1, Paul J. Wolters2§, and Joseph R. Arron1§ 1Genentech Research and Early Development, South San Francisco, CA 2Department of Medicine, University of California, San Francisco, CA 3Department of Surgery, University of California, San Francisco, CA ⌘Current address: Novartis Institutes for Biomedical Research, Emeryville, CA. #Current address: Gilead Sciences, Foster City, CA. *DJD and SC contributed equally to this manuscript §PJW and JRA co-directed this project Address correspondence to Paul J. Wolters, MD University of California, San Francisco Department of Medicine Box 0111 San Francisco, CA 94143-0111 [email protected] or Joseph R. Arron, MD, PhD Genentech, Inc. MS 231C 1 DNA Way South San Francisco, CA 94080 [email protected] 1 METHODS Human lung tissue samples Tissues were obtained at UCSF from clinical samples from IPF patients at the time of biopsy or lung transplantation. All patients were seen at UCSF and the diagnosis of IPF was established through multidisciplinary review of clinical, radiological, and pathological data according to criteria established by the consensus classification of the American Thoracic Society (ATS) and European Respiratory Society (ERS), Japanese Respiratory Society (JRS), and the Latin American Thoracic Association (ALAT) (ref. 5 in main text). Non-diseased normal lung tissues were procured from lungs not used by the Northern California Transplant Donor Network.
    [Show full text]
  • G Protein Alpha Inhibitor 1 (GNAI1) Rabbit Polyclonal Antibody 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 TA335036 G protein alpha inhibitor 1 (GNAI1) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: WB Recommended Dilution: WB Reactivity: Human, Mouse, Rat Host: Rabbit Isotype: IgG Clonality: Polyclonal Immunogen: The immunogen for anti-GNAI1 antibody: synthetic peptide directed towards the middle region of human GNAI1. Synthetic peptide located within the following region: YQLNDSAAYYLNDLDRIAQPNYIPTQQDVLRTRVKTTGIVETHFTFKDLH Formulation: Liquid. Purified antibody supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose. Note that this product is shipped as lyophilized powder to China customers. Purification: Affinity Purified Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 40 kDa Gene Name: G protein subunit alpha i1 Database Link: NP_002060 Entrez Gene 14677 MouseEntrez Gene 25686 RatEntrez Gene 2770 Human P63096 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 / 4 G protein alpha inhibitor 1 (GNAI1) Rabbit Polyclonal Antibody – TA335036 Background: Guanine nucleotide-binding proteins (G proteins) form a large family of signal-transducing molecules. They are found as heterotrimers made up of alpha, beta, and gamma subunits. Members of the G protein family have been characterized most extensively on the basis of the alpha subunit, which binds guanine nucleotide, is capable of hydrolyzing GTP, and interacts with specific receptor and effector molecules.
    [Show full text]