DOCSLIB.ORG

Wnt/Β-Catenin Signaling Promotes Self-Renewal and Inhibits the Primed State Transition in Naïve Human Embryonic Stem Cells

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Wnt/Β-Catenin Signaling Promotes Self-Renewal and Inhibits the Primed State Transition in Naïve Human Embryonic Stem Cells Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells Zhuojin Xua,b, Aaron M. Robitailleb,c, Jason D. Berndta,b,c, Kathryn C. Davidsond, Karin A. Fischerb,e, Julie Mathieub,e, Jennifer C. Pottera,b, Hannele Ruohola-Bakerb,e, and Randall T. Moona,b,c,1 aHoward Hughes Medical Institute, University of Washington School of Medicine, Seattle, WA 98109; bInstitute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA 98109; cDepartment of Pharmacology, University of Washington School of Medicine, Seattle, WA 98109; dAustralian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia; and eDepartment of Biochemistry, University of Washington School of Medicine, Seattle, WA 98109 Contributed by Randall T. Moon, August 24, 2016 (sent for review January 13, 2016; reviewed by Kouichi Hasegawa and Renee A. Reijo Pera) In both mice and humans, pluripotent stem cells (PSCs) exist in at least primed state (e.g., LIS1, WIN1, and H1-4iLIF lines) (3, 11–13)orby two distinct states of pluripotency, known as the naïve and primed deriving new hESC lines from human six- to eight-cell embryos (e.g., states. Our understanding of the intrinsic and extrinsic factors that ELF1 line) using naïve-state growth conditions (12). The molecular enable PSCs to self-renew and to transition between different plurip- programs used to support naïve pluripotency in hESCs and the de- otent states is important for understanding early development. In gree of conservation of the different states of pluripotency between mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC human and mouse PSCs is an area of active investigation (14). self-renewal and support the naïve state. In human embryonic stem Wnt/β-catenin signaling is a key signaling pathway involved in cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs embryonic development and is tightly regulated in PSCs (15–17). and is reduced or absent in primed-state hESCs. However, the role Wnt ligands are evolutionarily conserved secreted glycoproteins of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. involved in autocrine and paracrine cell signaling. In the absence Here, we demonstrate that inhibition of the secretion of Wnts or of Wnts, a “destruction complex” consisting of the cytosolic pro- β inhibition of the stabilization of -catenin in naïve hESCs reduces cell teins glycogen synthase kinase 3 (GSK3), AXIN, adenomatous proliferation and colony formation. Moreover, we show that addition polyposis coli (APC) protein, and casein kinase 1α phosphorylates of recombinant Wnt3a partially rescues cell proliferation in naïve β-catenin (encoded by the CTNNB1 gene). This pool of phos- hESCs caused by inhibition of Wnt secretion. Notably, inhibition of β β phorylated -catenin is ubiquitylated and thereby targeted to the Wnt/ -catenin signaling in naïve hESCs did not cause differentiation. proteasome for degradation. In the presence of Wnt ligands, Instead, it induced primed hESC-like proteomic and metabolic profiles. binding of Wnts to a heteromeric receptor complex leads to in- Thus, our results suggest that naïve hESCs secrete Wnts that activate hibition of the destruction complex, thus enabling β-catenin to autocrine or paracrine Wnt/β-catenin signaling to promote efficient accumulate in the cytoplasm. β-catenin translocates to the nucleus, self-renewal and inhibit the transition to the primed state. where it acts as a transcriptional coactivator for the T-cell factor Wnt/β-catenin signaling | naïve pluripotency | naïve-to-primed transition | (TCF) and lymphoid enhancing factor (LEF) family of DNA- human embryonic stem cells | self-renewal binding transcription factors (18). Wnt/β-catenin signaling is important in mESCs (19–22). Activation of Wnt/β-catenin signaling by recombinant Wnt3a or by inhibition luripotent stem cells (PSCs) undergo unlimited self-renewal of GSK3 synergizes with activation of JAK/STAT signaling by Pand can differentiate into cells representative of all three recombinant leukemia inhibitory factor (Lif) to promote self-renewal germ layers of the adult body (1). In both mice and humans, PSCs exist in at least two different states of pluripotency in vitro, commonly referred to as naïve and primed states. The different Significance states of pluripotency likely reflect different stages of embryonic development in vivo (2–5). Understanding the molecular un- Pluripotent stem cells (PSCs) can exist in a naïve or primed pluri- derpinnings of PSC behaviors in different pluripotency states and potency state. Naïve PSCs correspond to an earlier developmental identifying the molecular factors that control transitions between state more closely related to cells from the preimplantation em- these states may provide insight into early embryonic develop- bryo and may have more robust developmental potential than ment and contribute to efforts designed to use PSCs and their primed PSCs. However, understanding the molecular mechanisms derivatives for research and treatment of human diseases. that regulate naïve PSC behaviors such as self-renewal, differen- Much of our knowledge about the differences between naïve tiation, or preservation of the naïve state is incomplete. Here, we and primed states comes from research on mouse PSCs. Naïve report that Wnt/β-catenin signaling promotes the self-renewal of mouse embryonic stem cells (mESCs) are derived from embryonic naïve human embryonic stem cells (hESCs). When grown in con- cells of the preimplantation blastocyst and have a broad and ro- ditions that inhibit Wnt/β-catenin signaling, naïve hESCs remain bust developmental capacity (6, 7). In contrast, primed PSCs, undifferentiated but have a more primed-like protein expression β exemplified by mouse epiblast stem cells (mEpiSCs), display bia- profile. Our results suggest that Wnt/ -catenin signaling plays a ses toward lineage-specific differentiation (8). For example, al- critical role in regulating human naïve pluripotency. though mESCs can be used to generate a whole animal through Author contributions: Z.X. and J.D.B. designed research; Z.X., A.M.R., K.A.F., J.M., and J.C.P. tetraploid complementation when injected into a preimplantation performed research; J.C.P. assisted in cell culture; Z.X., A.M.R., J.D.B., K.C.D., H.R.-B., and R.T.M. blastocyst (9), mEpiSCs exhibit a poor capacity to contribute analyzed data; and Z.X., A.M.R., J.D.B., K.C.D., H.R.-B., and R.T.M. wrote the paper. to such chimeras. Conventional human embryonic stem cells Reviewers: K.H., Kyoto University; and R.A.R.P., Montana State University. (hESCs) exist in the primed state and exhibit marked similarities The authors declare no conflict of interest. in morphological, molecular, and epigenetic characteristics com- 1To whom correspondence should be addressed. Email: [email protected]. pared with mEpiSCs (10). More recently, naïve-state hESCs have This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. been generated by toggling conventional hESCs back from the 1073/pnas.1613849113/-/DCSupplemental. E6382–E6390 | PNAS | Published online October 3, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1613849113 Downloaded by guest on September 30, 2021 and inhibit spontaneous differentiation (19, 20). Dual inhibition of using the small molecules XAV939, which promotes degradation PNAS PLUS kinases GSK3 and MEK drives self-renewal and inhibits differ- of β-catenin (25), or IWP2, which blocks the secretion of Wnt li- entiation of naïve mESCs (2). Moreover, paracrine and autocrine gands (26), or by siRNA-mediated knockdown of CTNNB1 (13). Wnt signaling prevent naïve mESCs from converting into a primed We also found that the expression of genes involved in Wnt sig- mEpiSC-like state (22). naling pathways changes early during the naïve-to-primed transi- Whether Wnt/β-catenin signaling plays a similar role in naïve tion in hESCs (13). Nevertheless, we did not establish a causal link hESCs has not been fully investigated. Notably, naïve hESC lines between Wnt/β-catenin signaling and naïve hESC behaviors, such generated without transgenes (3, 12, 23, 24), including ELF1 and as self-renewal, differentiation, or preservation of the naïve state. H1-4iLIF hESCs, were derived and subsequently maintained in the Here, we show that Wnt/β-catenin signaling promotes self- presence of GSK3 inhibitors—a condition that may promote Wnt/ renewal of naïve hESCs but is dispensable for the maintenance β-catenin signaling. Recently, we reported that activation of a of pluripotency marker expression. Moreover, inhibition of Wnt/ β-catenin–activated reporter (BAR) is increased when ELF1 β-catenin signaling in naïve hESCs induces a more primed-like hESCs are grown in naïve conditions compared with primed con- global protein expression profile. Taken together, our results ditions (13). Moreover, the activity of BAR in naïve-state ELF1 implicate Wnt/β-catenin signaling as a positive regulator of hu- hESCs is suppressed by inhibition of Wnt/β-catenin signaling (13), man naïve pluripotency. A B AXIN2 TROY with CHIR w/o CHIR 1.2 naïve 0.8 primed 0.4 normalized Venus to naïve cells * 0.0 * 1.2 DMSO C w/o CHIR 0.8 XAV with CHIR 5 5 ** 10 86.7±5.3% 10 DMSO 0.4 * IWP2 90.3±4.0%0% 4 * 10 4 normalized to 10 0.0 CD9 Fold change 3 3 10 10 3.0 Ctrl si 2 2 * 10 10 0 CTNNB1 2 0 2.0 si -10 2 3 4 5 -10 3 4 5 1.0 AXIN1/2 0 10 10 10 0 10 10 10 Ctrl siRNA si normalized to * 0.0 * * Tra1-60 D d1-d3: DMSO XAV XAV IWP2 IWP2 d4-d6: DMSO XAV DMSO IWP2 DMSO Venus 250 120 120 50 150 200 90 40 33.4% 15.1% 24.9% 90 8.37% 20.2% 150 100 with CHIR 30 60 60 100 20 50 30 30 count 50 10 0 0 0 0 0 3 3 4 5 3 3 4 5 3 3 4 5 3 3 4 5 -103 0 103 104 105 0-10 10 10 10 -10 0 10 10 10 -10 0 10 10 10 -10 0 10 10 10 Venus 20 150 200 100 60 15 80 37.4% 150 16.6% 100 28.2% 6.64% 17.9% 10 40 60 100 w/o CHIR 40 50 count 5.0 20 50 20 0 0 0 0 0 3 3 4 5 3 3 4 5 3 3 4 5 0-10 10 10 10 0-10 10 10 10 -103 0 103 104 105 -103 0 103 104 105 -10 0 10 10 10 Fig.
Load more

Recommended publications
  • DF6216-CSRP1 Antibody
    Affinity Biosciences website:www.affbiotech.com order:[email protected] CSRP1 Antibody Cat.#: DF6216 Concn.: 1mg/ml Mol.Wt.: 21kDa Size: 50ul,100ul,200ul Source: Rabbit Clonality: Polyclonal Application: WB 1:500-1:2000, IHC 1:50-1:200, ELISA(peptide) 1:20000-1:40000 *The optimal dilutions should be determined by the end user. Reactivity: Human,Mouse,Rat Purification: The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific). Specificity: CSRP1 Antibody detects endogenous levels of total CSRP1. Immunogen: A synthesized peptide derived from human CSRP1, corresponding to a region within the internal amino acids. Uniprot: P21291 Description: This gene encodes a member of the cysteine-rich protein (CSRP) family. This gene family includes a group of LIM domain proteins, which may be involved in regulatory processes important for development and cellular differentiation. The LIM/double zinc-finger motif found in this gene product occurs in proteins with critical functions in gene regulation, cell growth, and somatic differentiation. Alternatively spliced transcript variants have been described. [provided by RefSeq, Aug 2010] Storage Condition and Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM Buffer: NaCl, 0.02% sodium azide and 50% glycerol.Store at -20 °C.Stable for 12 months from date of receipt. Western blot analysis of CSRP1 expression in Mouse lung lysate 1 / 2 Affinity Biosciences website:www.affbiotech.com order:[email protected] DF6216 at 1/100 staining Mouse brain tissue by IHC-P. The sample was formaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed.
    [Show full text]
  • Genome-Wide Association Study for Circulating Fibroblast Growth Factor
    www.nature.com/scientificreports OPEN Genome‑wide association study for circulating fbroblast growth factor 21 and 23 Gwo‑Tsann Chuang1,2,14, Pi‑Hua Liu3,4,14, Tsui‑Wei Chyan5, Chen‑Hao Huang5, Yu‑Yao Huang4,6, Chia‑Hung Lin4,7, Jou‑Wei Lin8, Chih‑Neng Hsu8, Ru‑Yi Tsai8, Meng‑Lun Hsieh9, Hsiao‑Lin Lee9, Wei‑shun Yang2,9, Cassianne Robinson‑Cohen10, Chia‑Ni Hsiung11, Chen‑Yang Shen12,13 & Yi‑Cheng Chang2,9,12* Fibroblast growth factors (FGFs) 21 and 23 are recently identifed hormones regulating metabolism of glucose, lipid, phosphate and vitamin D. Here we conducted a genome‑wide association study (GWAS) for circulating FGF21 and FGF23 concentrations to identify their genetic determinants. We enrolled 5,000 participants from Taiwan Biobank for this GWAS. After excluding participants with diabetes mellitus and quality control, association of single nucleotide polymorphisms (SNPs) with log‑transformed FGF21 and FGF23 serum concentrations adjusted for age, sex and principal components of ancestry were analyzed. A second model additionally adjusted for body mass index (BMI) and a third model additionally adjusted for BMI and estimated glomerular fltration rate (eGFR) were used. A total of 4,201 participants underwent GWAS analysis. rs67327215, located within RGS6 (a gene involved in fatty acid synthesis), and two other SNPs (rs12565114 and rs9520257, located between PHC2-ZSCAN20 and ARGLU1-FAM155A respectively) showed suggestive associations with serum FGF21 level (P = 6.66 × 10–7, 6.00 × 10–7 and 6.11 × 10–7 respectively). The SNPs rs17111495 and rs17843626 were signifcantly associated with FGF23 level, with the former near PCSK9 gene and the latter near HLA-DQA1 gene (P = 1.04 × 10–10 and 1.80 × 10–8 respectively).
    [Show full text]
  • Controls Homeostatic Splicing of ARGLU1 Mrna Stephan P
    Published online 28 November 2016 Nucleic Acids Research, 2017, Vol. 45, No. 6 3473–3486 doi: 10.1093/nar/gkw1140 An Ultraconserved Element (UCE) controls homeostatic splicing of ARGLU1 mRNA Stephan P. Pirnie, Ahmad Osman, Yinzhou Zhu and Gordon G. Carmichael* Department of Genetics and Genome Sciences, UCONN Health Center, 400 Farmington Avenue, Farmington, CT 06030, USA Received January 13, 2016; Revised October 25, 2016; Editorial Decision October 28, 2016; Accepted October 31, 2016 ABSTRACT or inhibit the usage of a particular splice site (1,2). The ex- pression of trans-acting factors in a developmental and tis- Arginine and Glutamate-Rich protein 1 (ARGLU1) is sue specific manner results in regulated splicing that isof- a protein whose function is poorly understood, but ten cell specific. Most notably, trans-acting proteins such as may act in both transcription and pre-mRNA splicing. the NOVA (3–6), the RBFOX (7) and SR protein (8–11) We demonstrate that the ARGLU1 gene expresses families, and a number of hnRNP (12,13) proteins compete at least three distinct RNA splice isoforms – a fully to bind nascent RNAs at specific motifs and drive regula- spliced isoform coding for the protein, an isoform tion of alternative splicing in a tissue and developmentally containing a retained intron that is detained in the specific manner. Alternative splicing is an important pro- nucleus, and an isoform containing an alternative cess that is seen in at least 95% of multi-exon genes in the exon that targets the transcript for nonsense medi- human transcriptome (14). Furthermore, alternative splic- ated decay.
    [Show full text]
  • Reconstructing Cell Cycle Pseudo Time-Series Via Single-Cell Transcriptome Data—Supplement
    School of Natural Sciences and Mathematics Reconstructing Cell Cycle Pseudo Time-Series Via Single-Cell Transcriptome Data—Supplement UT Dallas Author(s): Michael Q. Zhang Rights: CC BY 4.0 (Attribution) ©2017 The Authors Citation: Liu, Zehua, Huazhe Lou, Kaikun Xie, Hao Wang, et al. 2017. "Reconstructing cell cycle pseudo time-series via single-cell transcriptome data." Nature Communications 8, doi:10.1038/s41467-017-00039-z This document is being made freely available by the Eugene McDermott Library of the University of Texas at Dallas with permission of the copyright owner. All rights are reserved under United States copyright law unless specified otherwise. File name: Supplementary Information Description: Supplementary figures, supplementary tables, supplementary notes, supplementary methods and supplementary references. CCNE1 CCNE1 CCNE1 CCNE1 36 40 32 34 32 35 30 32 28 30 30 28 28 26 24 25 Normalized Expression Normalized Expression Normalized Expression Normalized Expression 26 G1 S G2/M G1 S G2/M G1 S G2/M G1 S G2/M Cell Cycle Stage Cell Cycle Stage Cell Cycle Stage Cell Cycle Stage CCNE1 CCNE1 CCNE1 CCNE1 40 32 40 40 35 30 38 30 30 28 36 25 26 20 20 34 Normalized Expression Normalized Expression Normalized Expression 24 Normalized Expression G1 S G2/M G1 S G2/M G1 S G2/M G1 S G2/M Cell Cycle Stage Cell Cycle Stage Cell Cycle Stage Cell Cycle Stage Supplementary Figure 1 | High stochasticity of single-cell gene expression means, as demonstrated by relative expression levels of gene Ccne1 using the mESC-SMARTer data. For every panel, 20 sample cells were randomly selected for each of the three stages, followed by plotting the mean expression levels at each stage.
    [Show full text]
  • S41467-020-18249-3.Pdf
    ARTICLE https://doi.org/10.1038/s41467-020-18249-3 OPEN Pharmacologically reversible zonation-dependent endothelial cell transcriptomic changes with neurodegenerative disease associations in the aged brain Lei Zhao1,2,17, Zhongqi Li 1,2,17, Joaquim S. L. Vong2,3,17, Xinyi Chen1,2, Hei-Ming Lai1,2,4,5,6, Leo Y. C. Yan1,2, Junzhe Huang1,2, Samuel K. H. Sy1,2,7, Xiaoyu Tian 8, Yu Huang 8, Ho Yin Edwin Chan5,9, Hon-Cheong So6,8, ✉ ✉ Wai-Lung Ng 10, Yamei Tang11, Wei-Jye Lin12,13, Vincent C. T. Mok1,5,6,14,15 &HoKo 1,2,4,5,6,8,14,16 1234567890():,; The molecular signatures of cells in the brain have been revealed in unprecedented detail, yet the ageing-associated genome-wide expression changes that may contribute to neurovas- cular dysfunction in neurodegenerative diseases remain elusive. Here, we report zonation- dependent transcriptomic changes in aged mouse brain endothelial cells (ECs), which pro- minently implicate altered immune/cytokine signaling in ECs of all vascular segments, and functional changes impacting the blood–brain barrier (BBB) and glucose/energy metabolism especially in capillary ECs (capECs). An overrepresentation of Alzheimer disease (AD) GWAS genes is evident among the human orthologs of the differentially expressed genes of aged capECs, while comparative analysis revealed a subset of concordantly downregulated, functionally important genes in human AD brains. Treatment with exenatide, a glucagon-like peptide-1 receptor agonist, strongly reverses aged mouse brain EC transcriptomic changes and BBB leakage, with associated attenuation of microglial priming. We thus revealed tran- scriptomic alterations underlying brain EC ageing that are complex yet pharmacologically reversible.
    [Show full text]
  • Screening and Identification of Hub Genes in Bladder Cancer by Bioinformatics Analysis and KIF11 Is a Potential Prognostic Biomarker
    ONCOLOGY LETTERS 21: 205, 2021 Screening and identification of hub genes in bladder cancer by bioinformatics analysis and KIF11 is a potential prognostic biomarker XIAO‑CONG MO1,2*, ZI‑TONG ZHANG1,3*, MENG‑JIA SONG1,2, ZI‑QI ZHOU1,2, JIAN‑XIONG ZENG1,2, YU‑FEI DU1,2, FENG‑ZE SUN1,2, JIE‑YING YANG1,2, JUN‑YI HE1,2, YUE HUANG1,2, JIAN‑CHUAN XIA1,2 and DE‑SHENG WENG1,2 1State Key Laboratory of Oncology in South China, Collaborative Innovation Centre for Cancer Medicine; 2Department of Biotherapy, Sun Yat‑Sen University Cancer Center; 3Department of Radiation Oncology, Sun Yat‑Sen University Cancer Center, Guangzhou, Guangdong 510060, P.R. China Received July 31, 2020; Accepted December 18, 2020 DOI: 10.3892/ol.2021.12466 Abstract. Bladder cancer (BC) is the ninth most common immunohistochemistry and western blotting. In summary, lethal malignancy worldwide. Great efforts have been devoted KIF11 was significantly upregulated in BC and might act as to clarify the pathogenesis of BC, but the underlying molecular a potential prognostic biomarker. The present identification mechanisms remain unclear. To screen for the genes associated of DEGs and hub genes in BC may provide novel insight for with the progression and carcinogenesis of BC, three datasets investigating the molecular mechanisms of BC. were obtained from the Gene Expression Omnibus. A total of 37 tumor and 16 non‑cancerous samples were analyzed to Introduction identify differentially expressed genes (DEGs). Subsequently, 141 genes were identified, including 55 upregulated and Bladder cancer (BC) is the ninth most common malignancy 86 downregulated genes. The protein‑protein interaction worldwide with substantial morbidity and mortality.
    [Show full text]
  • Methods in and Applications of the Sequencing of Short Non-Coding Rnas" (2013)
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2013 Methods in and Applications of the Sequencing of Short Non- Coding RNAs Paul Ryvkin University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Bioinformatics Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation Ryvkin, Paul, "Methods in and Applications of the Sequencing of Short Non-Coding RNAs" (2013). Publicly Accessible Penn Dissertations. 922. https://repository.upenn.edu/edissertations/922 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/922 For more information, please contact [email protected]. Methods in and Applications of the Sequencing of Short Non-Coding RNAs Abstract Short non-coding RNAs are important for all domains of life. With the advent of modern molecular biology their applicability to medicine has become apparent in settings ranging from diagonistic biomarkers to therapeutics and fields angingr from oncology to neurology. In addition, a critical, recent technological development is high-throughput sequencing of nucleic acids. The convergence of modern biotechnology with developments in RNA biology presents opportunities in both basic research and medical settings. Here I present two novel methods for leveraging high-throughput sequencing in the study of short non- coding RNAs, as well as a study in which they are applied to Alzheimer's Disease (AD). The computational methods presented here include High-throughput Annotation of Modified Ribonucleotides (HAMR), which enables researchers to detect post-transcriptional covalent modifications ot RNAs in a high-throughput manner. In addition, I describe Classification of RNAs by Analysis of Length (CoRAL), a computational method that allows researchers to characterize the pathways responsible for short non-coding RNA biogenesis.
    [Show full text]
  • CRIP1 Antibody (C-Term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # Ap4707b
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 CRIP1 Antibody (C-term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP4707b Specification CRIP1 Antibody (C-term) - Product Information Application WB, IHC-P, FC,E Primary Accession P50238 Other Accession P63255, P63254, Q56K04 Reactivity Human Predicted Bovine, Mouse, Rat Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Calculated MW 8533 Antigen Region 48-77 CRIP1 Antibody (C-term) - Additional Information Gene ID 1396 All lanes : Anti-CRIP1 Antibody (C-term) at 1:8000 dilution Lane 1: T47D whole cell Other Names lysate Lane 2: THP-1 whole cell lysate Cysteine-rich protein 1, CRP-1, Cysteine-rich Lysates/proteins at 20 µg per lane. heart protein, CRHP, hCRHP, Cysteine-rich Secondary Goat Anti-Rabbit IgG, (H+L), intestinal protein, CRIP, CRIP1, CRIP, CRP1 Peroxidase conjugated at 1/10000 dilution. Predicted band size : 9 kDa Blocking/Dilution Target/Specificity buffer: 5% NFDM/TBST. This CRIP1 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 48-77 amino acids from the C-terminal region of human CRIP1. Dilution WB~~1:8000 IHC-P~~1:50~100 FC~~1:10~50 Format Purified polyclonal antibody supplied in PBS with 0.09% (W/V) sodium azide. This antibody is purified through a protein A column, followed by peptide affinity CRIP1 Antibody (C-term) (Cat. #AP4707b) purification. IHC analysis in formalin fixed and paraffin embedded bladder carcinoma followed by Storage peroxidase conjugation of the secondary Maintain refrigerated at 2-8°C for up to 2 antibody and DAB staining.
    [Show full text]
  • Identification of Differentially Expressed Genes in Human Bladder Cancer Through Genome-Wide Gene Expression Profiling
    521-531 24/7/06 18:28 Page 521 ONCOLOGY REPORTS 16: 521-531, 2006 521 Identification of differentially expressed genes in human bladder cancer through genome-wide gene expression profiling KAZUMORI KAWAKAMI1,3, HIDEKI ENOKIDA1, TOKUSHI TACHIWADA1, TAKENARI GOTANDA1, KENGO TSUNEYOSHI1, HIROYUKI KUBO1, KENRYU NISHIYAMA1, MASAKI TAKIGUCHI2, MASAYUKI NAKAGAWA1 and NAOHIKO SEKI3 1Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520; Departments of 2Biochemistry and Genetics, and 3Functional Genomics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Received February 15, 2006; Accepted April 27, 2006 Abstract. Large-scale gene expression profiling is an effective CKS2 gene not only as a potential biomarker for diagnosing, strategy for understanding the progression of bladder cancer but also for staging human BC. This is the first report (BC). The aim of this study was to identify genes that are demonstrating that CKS2 expression is strongly correlated expressed differently in the course of BC progression and to with the progression of human BC. establish new biomarkers for BC. Specimens from 21 patients with pathologically confirmed superficial (n=10) or Introduction invasive (n=11) BC and 4 normal bladder samples were studied; samples from 14 of the 21 BC samples were subjected Bladder cancer (BC) is among the 5 most common to microarray analysis. The validity of the microarray results malignancies worldwide, and the 2nd most common tumor of was verified by real-time RT-PCR. Of the 136 up-regulated the genitourinary tract and the 2nd most common cause of genes we detected, 21 were present in all 14 BCs examined death in patients with cancer of the urinary tract (1-7).
    [Show full text]
  • Genomic and Transcriptomic Investigations Into the Feed Efficiency Phenotype of Beef Cattle
    Provided by the author(s) and NUI Galway in accordance with publisher policies. Please cite the published version when available. Title Genomic and transcriptomic investigations into the feed efficiency phenotype of beef cattle Author(s) Higgins, Marc Publication Date 2019-03-06 Publisher NUI Galway Item record http://hdl.handle.net/10379/15008 Downloaded 2021-09-25T18:07:39Z Some rights reserved. For more information, please see the item record link above. Genomic and Transcriptomic Investigations into the Feed Efficiency Phenotype of Beef Cattle Marc Higgins, B.Sc., M.Sc. A thesis submitted for the Degree of Doctor of Philosophy to the Discipline of Biochemistry, School of Natural Sciences, National University of Ireland, Galway. Supervisor: Dr. Derek Morris Discipline of Biochemistry, School of Natural Sciences, National University of Ireland, Galway. Supervisor: Dr. Sinéad Waters Teagasc, Animal and Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Grange. Submitted November 2018 Table of Contents Declaration ................................................................................................................ vii Funding .................................................................................................................... viii Acknowledgements .................................................................................................... ix Abstract ......................................................................................................................
    [Show full text]
  • A Sonic Hedgehog Signaling Domain in the Arterial Adventitia Supports Resident Sca1؉ Smooth Muscle Progenitor Cells
    A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1؉ smooth muscle progenitor cells Jenna N. Passman*†, Xiu Rong Dong*‡, San-Pin Wu*§, Colin T. Maguire*‡, Kelly A. Hogan¶, Victoria L. Bautch*†¶, and Mark W. Majesky*†‡ʈ** *Carolina Cardiovascular Biology Center, Departments of ‡Medicine, ʈGenetics, and ¶Biology, and †Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599 Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 22, 2008 (received for review December 6, 2007) We characterize a sonic hedgehog (Shh) signaling domain re- interstitial fibroblasts (13, 14), and promotes endothelial cell stricted to the adventitial layer of artery wall that supports resi- chemotaxis and tube formation (17, 18). Therefore, Shh plays dent Sca1-positive vascular progenitor cells (AdvSca1). Using important roles in cell–cell communication during vascular patched-1 (Ptc1lacZ) and patched-2 (Ptc2lacZ) reporter mice, adven- development and adult wound repair. titial Shh signaling activity was first detected at embryonic day (E) Hedgehog (Hh) proteins signal by binding to patched-1 (Ptc1) 15.5, reached the highest levels between postnatal day 1 (P1) and or patched-2 (Ptc2) receptors and cdo/boc accessory proteins at P10, was diminished in adult vessels, and colocalized with a the cell surface, resulting in derepression of smoothened (smo) circumferential ring of Shh protein deposited between the media (19). Ptc proteins are 12-transmembrane domain receptors that ؊/؊ and adventitia. In Shh mice, AdvSca1 cells normally found at the are structurally related to the resistance-nodulation-division aortic root were either absent or greatly diminished in number.
    [Show full text]
  • BMC Developmental Biology Biomed Central
    BMC Developmental Biology BioMed Central Research article Open Access Targeted disruption of the mouse Csrp2 gene encoding the cysteine- and glycine-rich LIM domain protein CRP2 result in subtle alteration of cardiac ultrastructure Julia F Sagave1, Markus Moser2, Elisabeth Ehler3, Sabine Weiskirchen1, Doris Stoll1, Kalle Günther4, Reinhard Büttner5 and Ralf Weiskirchen*1 Address: 1Institute of Clinical Chemistry and Pathobiochemistry, RWTH- University Hospital Aachen, Germany, 2Max Planck-Institute for Biochemistry, Martinsried, Germany, 3The Randall Division of Cell and Molecular Biophysics and The Cardiovascular Division, King's College London, UK, 4Qiagen, Hilden, Germany and 5Institute of Pathology, University Hospital Bonn, Germany Email: Julia F Sagave - [email protected]; Markus Moser - [email protected]; Elisabeth Ehler - [email protected]; Sabine Weiskirchen - [email protected]; Doris Stoll - [email protected]; Kalle Günther - [email protected]; Reinhard Büttner - [email protected]; Ralf Weiskirchen* - [email protected] * Corresponding author Published: 19 August 2008 Received: 19 March 2008 Accepted: 19 August 2008 BMC Developmental Biology 2008, 8:80 doi:10.1186/1471-213X-8-80 This article is available from: http://www.biomedcentral.com/1471-213X/8/80 © 2008 Sagave et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The cysteine and glycine rich protein 2 (CRP2) encoded by the Csrp2 gene is a LIM domain protein expressed in the vascular system, particularly in smooth muscle cells.
    [Show full text]