DOCSLIB.ORG

SKA2 Mediates Invasion and Metastasis in Human Breast Cancer Via EMT

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SKA2 Mediates Invasion and Metastasis in Human Breast Cancer Via EMT MOLECULAR MEDICINE REPORTS 19: 515-523, 2019 SKA2 mediates invasion and metastasis in human breast cancer via EMT ZHOUHUI REN1,2*, TONG YANG2*, PINGPING ZHANG3, KAITAI LIU4, WEIHONG LIU1 and PING WANG1 1Zhejiang Provincial Key Laboratory of Pathophysiology, Ningbo University School of Medicine, Ningbo, Zhejiang 315211; 2Department of Oncology Surgery, Ningbo No. 2 Hospital, Ningbo, Zhejiang 315010; 3Department of Gynaecology, Ningbo Women and Children's Hospital, Ningbo, Zhejiang 315012; 4Department of Oncology, Ningbo Medical Center, Li Huili Hospital, Ningbo, Zhejiang 315041, P.R. China Received August 13, 2017; Accepted October 2, 2018 DOI: 10.3892/mmr.2018.9623 Abstract. Spindle and kinetochore-associated protein 2 These results demonstrated that SKA2 may be associated with (SKA2) is essential for regulating the progression of mitosis. In breast cancer metastasis, and siSKA2 inhibited the invasion recent years, SKA2 upregulation has been detected in various and metastasis of breast cancer via translocation of E-cadherin human malignancies and the role of SKA2 in tumorigenesis from the cytoplasm to the nucleus. has received increasing attention. However, the expression and functional significance of SKA2 in breast cancer are Introduction not completely understood. To study the effects of SKA2 on breast cancer, the expression levels of SKA2 in breast cancer Spindle and kinetochore-associated (SKA)2 is located on tissues and cell lines were evaluated by western blotting, chromosome 17 of the human genome and has been identi- reverse transcription-quantitative polymerase chain reaction fied as a conserved protein involved in the kinetochore and immunohistochemical staining. The results demonstrated complex (1,2). SKA2, together with its cofactors SKA1 and that SKA2 expression was increased in breast cancer tissues SKA3, constitute the SKA complex, which maintains the and cells, and SKA2 overexpression was associated with metaphase plate and/or spindle checkpoint silencing (3-5). clinical stage and lymph node metastasis. Functional investi- Checkpoint-dependent delays in a metaphase-like state are gations revealed that SKA2 knockdown in breast cancer cells prolonged by RNA interference-mediated SKA2 deple- significantly reduced migration and invasion, and resulted in tion (6,7). In addition, SKA2 has been reported to serve a the decreased expression levels of matrix metalloproteinase role in tumorigenesis. Aberrant patterns of SKA2 expression (MMP)2 and MMP9. Furthermore, the typical microtubule have been observed in several types of cancer, including lung arrangement was altered in SKA2 small interfering RNA cancer (8-10), kidney cancer (11), pancreatic cancer (12), gastric (siSKA2)-transfected cells. Reduced levels of SKA2 also cancer (13), glioma (14) and osteosarcoma (15,16). However, to downregulated the expression of epithelial-mesenchymal the best of our knowledge, the roles of SKA2 in breast cancer transition proteins, including fibronectin, N-cadherin and migration remain to be elucidated. vimentin, whereas there were no alterations in the protein Breast cancer is common in women worldwide. The first expression levels of E-cadherin. Conversely, upregulation highest incidence rates and second highest mortality rates of SKA2 decreased the expression levels of E-cadherin, have been demonstrated in the developed world (17,18). and increased N‑cadherin, fibronectin and vimentin levels. Furthermore, breast cancer is prone to metastasis and results Notably, it was demonstrated that E-cadherin was translocated in poor prognosis (19,20). Although marked progress has been from the cytoplasm to the nucleus in siSKA2-transfected cells. achieved in breast cancer therapy due to modern technology, unknown molecular mechanisms of metastasis remain and require further investigation. As a fundamental process of migration, epithelial-mesenchymal transition (EMT) serves a crucial role in breast cancer progression (21-24). However, the Correspondence to: Dr Ping Wang, Zhejiang Provincial Key specific regulatory mechanism mediating EMT and SKA2 in Laboratory of Pathophysiology, Ningbo University School of breast cancer progression remains to be investigated. In the Medicine, 818 Fenghua Road, Ningbo, Zhejiang 315211, P.R. China E-mail: [email protected] present study, it was demonstrated that SKA2 was upregulated in human �����������������������������������������������������breast����������������������������������������������� cancer cell lines and tissues����������������� by �������������reverse tran- *Contributed equally scription quantitative polymerase chain reaction (RT-qPCR) and western blotting. Small interfering (si)SKA2 was used to Key words: spindle and kinetochore-associated protein 2, breast knockdown the expression of SKA2 in MCF-7 and T47D cells, cancer, invasion, metastasis, epithelial-mesenchymal transition the results demonstrated that decreasing the level of SKA2 inhibited the metastasis of the breast cancer cells by wound healing assay and cell invasion assays. However, although the 516 REN et al: SKA2 MEDIATES INVASION AND METASTASIS IN HUMAN BREAST CANCER results are promising, further studies are needed to confirm 12% SDS-PAGE and were transferred onto polyvinylidende this. Therefore, the present study further investigated whether di�uoride membranes (EMD Millipore, Billerica, MA, USA) SKA2 regulates metastasis via EMT-associated proteins and for immunoblotting. After blocking with 5% bovine serum matrix metalloproteinase (MMP)-2/9. The current study albumin (BSA; Beijing Solarbio Science & Technology aimed to determine the potential role of SKA2 in breast cancer Co., Ltd., Beijing, China) for 2 h at room temperature, the invasion, and its molecular mechanism. membranes were incubated with specific primary antibodies diluted in 1X Tris-buffered saline-0.1% Tween-20 overnight Patients and methods at 4˚C. The following antibodies were used: SKA2 (1:1,�00; cat. no. ab91551), MMP2 (1:1,500; cat. no. ab7033), MMP9 Patients. Tumor specimens and adjacent tissues were collected (1:1,500; cat. no. ab137651), vimentin (1:2,000; cat. no. ab8978), from patients who were diagnosed with breast cancer fibronectin (1:2,000; cat. no. ab2413; all Abcam, Cambridge, (stages I-IV) and underwent surgery at Ningbo No. 2 Hospital UK), N-cadherin (1:2,000; cat. no. 14215), E-cadherin (Ningbo, China) between March 2015 and August 2017. None (1:2,000; cat. no. 3195), β-actin (1:1,500; cat. no. 8457), of these patients underwent local or systemic therapy prior to Histone H3 (1:1,500; cat. no. 9728) and GAPDH (1:1,500; surgery. The clinical characteristics of these 160 patients are cat. no. 5174; all Cell Signaling Technology, Inc., Danvers, described in Table I. The present study was approved by the MA, USA). Subsequently, membranes were incubated for 1 h Research Ethics Committee of Ningbo No. 2 Hospital, and all at room temperature with goat anti-rabbit immunoglobulin patients provided written informed consent. (Ig)G-horseradish peroxidase (HRP) or goat anti-mouse IgG-HRP (1:5,000; cat. nos. BA1054/BA1050; Wuhan Boster Cell lines and transfection. Human breast cancer cell lines Biological Technology, Ltd., Wuhan, China). The protein bands MCF-7, T47D and MDA-MB-231, and normal MCF-10A cells on the membrane were visualized using a chemiluminescence were purchased from the American Type Culture Collection imaging system (LI-COR Biosciences, Lincoln, CA, USA) (Manassas, VA, USA). All cells were cultured in Dulbecco's and detected by chemiluminescence. Densitometric analysis modified Eagle's medium (DMEM; HyClone; GE Healthcare was performed by Tanon GIS version 4.1.2 software (Tanon Life Sciences, Logan, UT, USA) supplemented with 10% fetal Science and Technology Co., Ltd., Shanghai, China). bovine serum (FBS; ExCell Bio, Shanghai, China) at 37˚C in an incubator containing 5% CO2. RT‑qPCR. The mRNA expression levels of SKA2 were Cells ���������������������������������������������������(��������������������������������������������������MCF-7 and T47D������������������������������������) �����������������������������������were transfected with small inter- detected using RT‑qPCR. Brie�y, total RNA was isolated fering RNA (si)SKA2 (sense, 5'-GGC UGG AAU AUG AAA UCA using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, ATT-3' and antisense, 5'-UUG AUU UCA UAU UCC AGC CTT-3'), Inc.) and 1 µg total RNA was subjected to first-strand si-negative control (NC; sense, 5'-UCC UCC GAA CGU GUC ACG cDNA synthesis for 1� min at 37˚C and � sec at 8�˚C using UTT-3' and antisense, 5'-ACG UGA CAC GUU CGG AGA ATT-3') a reverse transcription kit (Thermo Fisher Scientific, Inc.). (both Shanghai GenePharma Co., Ltd., Shanghai, China), cDNA was amplified by RT-qPCR using SYBR-Green SKA2cDNA plasmid������������ (����������Ref������� ������Seq: M�������������������������001100595.1�������������, Atgg����������� cctcg- PCR Master Mix (Roche Applied Science, Madison, WI, gaggt ggggcacaat ttggagtcgc cggaaactcc gcggcgga ggctggacca USA) on a LightCycler® 480 system (9�˚C/10 min/1 cycle, gagtcgagtt ctcctcct gcaccaaagg gagccgccac tctggtgt ctaaaccgcc 9�˚C/10 sec/45 cycles, 60˚C/60 sec/45 cycles; Roche Applied tcggttccag gctgagt ctgatctgga ttacattcaa caggctgg aatatgaaat Science) and fold-changes were calculated by relative quanti- caagactaat tcctgatt cagcaagtga gctgtcacca ctga) or NC plasmid fication (2-∆∆Cq) (25). The primers used were as follows: SKA2 (both Shanghai Genechem Co., Ltd., Shanghai, China) using forward, 5'-CTG AAA CTA TGC TAA GTG GGG GAG-3', Lipofectamine®
Load more

Recommended publications
  • Towards Personalized Medicine in Psychiatry: Focus on Suicide
    TOWARDS PERSONALIZED MEDICINE IN PSYCHIATRY: FOCUS ON SUICIDE Daniel F. Levey Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Program of Medical Neuroscience, Indiana University April 2017 ii Accepted by the Graduate Faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Andrew J. Saykin, Psy. D. - Chair ___________________________ Alan F. Breier, M.D. Doctoral Committee Gerry S. Oxford, Ph.D. December 13, 2016 Anantha Shekhar, M.D., Ph.D. Alexander B. Niculescu III, M.D., Ph.D. iii Dedication This work is dedicated to all those who suffer, whether their pain is physical or psychological. iv Acknowledgements The work I have done over the last several years would not have been possible without the contributions of many people. I first need to thank my terrific mentor and PI, Dr. Alexander Niculescu. He has continuously given me advice and opportunities over the years even as he has suffered through my many mistakes, and I greatly appreciate his patience. The incredible passion he brings to his work every single day has been inspirational. It has been an at times painful but often exhilarating 5 years. I need to thank Helen Le-Niculescu for being a wonderful colleague and mentor. I learned a lot about organization and presentation working alongside her, and her tireless work ethic was an excellent example for a new graduate student. I had the pleasure of working with a number of great people over the years. Mikias Ayalew showed me the ropes of the lab and began my understanding of the power of algorithms.
    [Show full text]
  • Global Identification of Hnrnp A1 Binding Sites for SSO-Based Splicing Modulation Gitte H
    Bruun et al. BMC Biology (2016) 14:54 DOI 10.1186/s12915-016-0279-9 RESEARCH ARTICLE Open Access Global identification of hnRNP A1 binding sites for SSO-based splicing modulation Gitte H. Bruun1, Thomas K. Doktor1, Jonas Borch-Jensen1, Akio Masuda2, Adrian R. Krainer3, Kinji Ohno2 and Brage S. Andresen1* Abstract Background: Many pathogenic genetic variants have been shown to disrupt mRNA splicing. Besides splice mutations in the well-conserved splice sites, mutations in splicing regulatory elements (SREs) may deregulate splicing and cause disease. A promising therapeutic approach is to compensate for this deregulation by blocking other SREs with splice-switching oligonucleotides (SSOs). However, the location and sequence of most SREs are not well known. Results: Here, we used individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) to establish an in vivo binding map for the key splicing regulatory factor hnRNP A1 and to generate an hnRNP A1 consensus binding motif. We find that hnRNP A1 binding in proximal introns may be important for repressing exons. We show that inclusion of the alternative cassette exon 3 in SKA2 can be significantly increased by SSO-based treatment which blocks an iCLIP-identified hnRNP A1 binding site immediately downstream of the 5’ splice site. Because pseudoexons are well suited as models for constitutive exons which have been inactivated by pathogenic mutations in SREs, we used a pseudoexon in MTRR as a model and showed that an iCLIP-identified hnRNP A1 binding site downstream of the 5′ splice site can be blocked by SSOs to activate the exon. Conclusions: The hnRNP A1 binding map can be used to identify potential targets for SSO-based therapy.
    [Show full text]
  • A Chromosome-Centric Human Proteome Project (C-HPP) To
    computational proteomics Laboratory for Computational Proteomics www.FenyoLab.org E-mail: [email protected] Facebook: NYUMC Computational Proteomics Laboratory Twitter: @CompProteomics Perspective pubs.acs.org/jpr A Chromosome-centric Human Proteome Project (C-HPP) to Characterize the Sets of Proteins Encoded in Chromosome 17 † ‡ § ∥ ‡ ⊥ Suli Liu, Hogune Im, Amos Bairoch, Massimo Cristofanilli, Rui Chen, Eric W. Deutsch, # ¶ △ ● § † Stephen Dalton, David Fenyo, Susan Fanayan,$ Chris Gates, , Pascale Gaudet, Marina Hincapie, ○ ■ △ ⬡ ‡ ⊥ ⬢ Samir Hanash, Hoguen Kim, Seul-Ki Jeong, Emma Lundberg, George Mias, Rajasree Menon, , ∥ □ △ # ⬡ ▲ † Zhaomei Mu, Edouard Nice, Young-Ki Paik, , Mathias Uhlen, Lance Wells, Shiaw-Lin Wu, † † † ‡ ⊥ ⬢ ⬡ Fangfei Yan, Fan Zhang, Yue Zhang, Michael Snyder, Gilbert S. Omenn, , Ronald C. Beavis, † # and William S. Hancock*, ,$, † Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States ‡ Stanford University, Palo Alto, California, United States § Swiss Institute of Bioinformatics (SIB) and University of Geneva, Geneva, Switzerland ∥ Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States ⊥ Institute for System Biology, Seattle, Washington, United States ¶ School of Medicine, New York University, New York, United States $Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia ○ MD Anderson Cancer Center, Houston, Texas, United States ■ Yonsei University College of Medicine, Yonsei University,
    [Show full text]
  • SKA2 Methylation Is Associated with Decreased Prefrontal Cortical
    SKA2 methylation is associated with decreased prefrontal cortical thickness and greater PTSD severity among trauma-exposed veterans Naomi Sadeh, VA Boston Healthcare System Jeffrey M. Spielberg, Boston University Mark W. Logue, VA Boston Healthcare System Erika J. Wolf, VA Boston Healthcare System Alicia K Smith, Emory University Joanna Lusk, VA Boston Healthcare System Jasmeet P. Hayes, VA Boston Healthcare System Emily Sperbeck, VA Boston Healthcare System William P. Milberg, VA Boston Healthcare System Regina E. McGlinchey, VA Boston Healthcare System Only first 10 authors above; see publication for full author list. Journal Title: Molecular Psychiatry Volume: Volume 21, Number 3 Publisher: Nature Publishing Group | 2016-03-01, Pages 357-363 Type of Work: Article | Post-print: After Peer Review Publisher DOI: 10.1038/mp.2015.134 Permanent URL: https://pid.emory.edu/ark:/25593/rptgj Final published version: http://dx.doi.org/10.1038/mp.2015.134 Copyright information: © 2016 Macmillan Publishers Limited. Accessed September 28, 2021 9:19 PM EDT HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Mol Psychiatry Manuscript Author . Author Manuscript Author manuscript; available in PMC 2016 May 18. Published in final edited form as: Mol Psychiatry. 2016 March ; 21(3): 357–363. doi:10.1038/mp.2015.134. SKA2 Methylation is associated with Decreased Prefrontal Cortical Thickness and Greater PTSD Severity among Trauma- Exposed Veterans Naomi Sadeh, Ph.D.1,3, Jeffrey M. Spielberg, Ph.D.2,3, Mark W. Logue, Ph.D.4, Erika J. Wolf, Ph.D.1,3, Alicia K. Smith, Ph.D.5, Joanna Lusk, B.A.1, Jasmeet P.
    [Show full text]
  • Genetic Variant in CACNA1C Is Associated with PTSD in Traumatized Police Officers
    European Journal of Human Genetics (2018) 26:247–257 https://doi.org/10.1038/s41431-017-0059-1 ARTICLE Genetic variant in CACNA1C is associated with PTSD in traumatized police officers 1 2,3 4 1 4 1 Izabela M. Krzyzewska ● Judith B.M. Ensink ● Laura Nawijn ● Adri N. Mul ● Saskia B. Koch ● Andrea Venema ● 1 4 5 2,3 4,6 Vinod Shankar ● Jessie L. Frijling ● Dirk J. Veltman ● Ramon J.L. Lindauer ● Miranda Olff ● 1 4 1 Marcel M.A.M. Mannens ● Mirjam van Zuiden ● Peter Henneman Received: 7 March 2017 / Revised: 6 October 2017 / Accepted: 31 October 2017 / Published online: 23 January 2018 © The Author(s) 2018. This article is published with open access Abstract Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder that may develop after a traumatic event. Here we aimed to identify epigenetic and genetic loci associated with PTSD. We included 73 traumatized police officers with extreme phenotypes regarding symptom severity despite similar trauma history: n = 34 had PTSD and n = 39 had minimal PTSD symptoms. Epigenetic and genetic profiles were based on the Illumina HumanMethylation450 BeadChip. We searched for differentially methylated probes (DMPs) and differentially methylated regions (DMRs). For genetic associations we fi 1234567890 analyzed the CpG-SNPs present on the array. We detected no genome-wide signi cant DMPs and we did not replicate previously reported DMPs associated with PTSD. However, GSE analysis of the top 100 DMPs showed enrichment of three genes involved in the dopaminergic neurogenesis pathway. Furthermore, we observed a suggestive association of one relatively large DMR between patients and controls, which was located at the PAX8 gene and previously associated with other psychiatric disorders.
    [Show full text]
  • By Makena L. Clive a Dissertation Submitted to Johns Hopkins University in Conformity with the Requirements for the Degree of Do
    UNDERSTANDING SUICIDE BIOMARKER SKA2: DISCOVERY OF A SUICIDE BIOSIGNATURE TO AUGMENT SKA2 SUICIDE PREDICTION AND INVESTIGATING SKA2 REGULATORY MECHANISMS by Makena L. Clive A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy. Baltimore, Maryland May, 2017 © 2017 Makena L. Clive All Rights Reserved Abstract….. Suicide is the 2nd leading cause of death for ages 10-34 in the United States. Suicide rates have risen dramatically since 1999, with an estimated 25 attempts per single completed suicide, calling for increased efforts to improve prevention strategies. Suicide is a state that is associated with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. The spindle and kinetochore associated complex 2 (SKA2) gene was recently discovered as a biomarker of suicide and HPA-axis dysregulation. Genetic and epigenetic variation at rs7208505, a single nucleotide polymorphism located in the 3’ untranslated region of SKA2, interacted with stress/anxiety metrics to predict suicidal behavior. Additionally, SKA2 was downregulated in the brains of suicide decedents. Little is known concerning the regulation and function of SKA2 in regards to HPA-axis dysregulation or suicide. The goal of this dissertation was to improve suicide prevention by enhancing our existing SKA2 suicide prediction model to better identify at-risk individuals, and to increase understanding of underlying suicide biology by investigating the regulation of SKA2. We used a custom bioinformatics approach to derive a DNA methylation biosignature that both interacted with rs7208505 methylation in post mortem prefrontal cortex and predicted suicide attempt in peripheral blood samples. Replacing stress/anxiety metrics in the SKA2 suicide prediction model with this biosignature improved prediction of suicidal behaviors.
    [Show full text]
  • Epigenetic Variation at SKA2 Predicts Suicide Phenotypes and Internalizing Psychopathology
    HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Depress Manuscript Author Anxiety. Author Manuscript Author manuscript; available in PMC 2017 April 01. Published in final edited form as: Depress Anxiety. 2016 April ; 33(4): 308–315. doi:10.1002/da.22480. Epigenetic Variation at SKA2 Predicts Suicide Phenotypes and Internalizing Psychopathology Naomi Sadeh1,2, Erika J. Wolf1,2, Mark W. Logue3, Jasmeet P. Hayes1,2, Annjanette Stone4, L. Michelle Griffin4, Steven A. Schichman4, and Mark W. Miller1,2 1 National Center for PTSD, Behavioral Science Division, VA Boston Healthcare System 2 Department of Psychiatry, Boston University School of Medicine 3 Research Service, VA Boston Healthcare System, Boston, MA; Biomedical Genetics, Boston University School of Medicine, Boston, MA; Department of Biostatistics, Boston University School of Public Health 4 Pharmacogenomics Analysis Laboratory, Research Service, Central Arkansas Veterans Healthcare System Abstract Background—"DNA methylation of the SKA2 gene has recently been implicated as a biomarker of suicide risk and posttraumatic stress disorder (PTSD). To examine the specificity and reliability of these findings, we examined associations between SKA2 DNA methylation, broad dimensions of psychiatric symptoms, and suicide phenotypes in adults with high levels of trauma exposure. Methods—A total of 466 White, non-Hispanic veterans and their intimate partners (65% male) underwent clinical assessment and had blood drawn for genotyping and methylation analysis. DNA methylation of the CpG locus cg13989295 and genotype at the methylation-associated single-nucleotide polymorphism (SNP) rs7208505 were examined in relation to current and lifetime PTSD, internalizing and externalizing psychopathology, and suicide phenotypes (ideation, plans, and attempts). Results—DNA methylation at the previously implicated SKA2 CpG locus (cg13989295) was associated with current and lifetime symptoms of internalizing (but not externalizing) disorders.
    [Show full text]
  • Rabbit Anti-SKA2/FITC Conjugated Antibody
    SunLong Biotech Co.,LTD Tel: 0086-571- 56623320 Fax:0086-571- 56623318 E-mail:[email protected] www.sunlongbiotech.com Rabbit Anti-SKA2/FITC Conjugated antibody SL7847R-FITC Product Name: Anti-SKA2/FITC Chinese Name: FITC标记的纺锤体和着丝粒相关蛋白2抗体 FAM33A; Family with sequence similarity 33, member A; FLJ12758; MGC110975; Protein FAM33A; SKA 2; SKA2; SKA2_HUMAN; Spindle and kinetochore associated Alias: complex subunit 2; Spindle and kinetochore associated protein 2; Spindle and kinetochore-associated protein 2; Spindle and KT (kinetochore) associated 2; Spindle and KT associated 2. Organism Species: Rabbit Clonality: Polyclonal React Species: Human,Mouse,Rat,Dog,Pig,Cow,Horse,Rabbit, IF=1:50-200 Applications: not yet tested in other applications. optimal dilutions/concentrations should be determined by the end user. Molecular weight: 14kDa Form: Lyophilized or Liquid Concentration: 1mg/ml immunogen: KLH conjugated synthetic peptide derived from human SKA2 Lsotype: IgGwww.sunlongbiotech.com Purification: affinity purified by Protein A Storage Buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.03% Proclin300 and 50% Glycerol. Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year Storage: when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. background: Ska2 (spindle and kinetochore associated complex subunit 2), also known as FAM33A, is a 121 amino acid component of the Ska1 complex, a microtubule-binding Product Detail: subcomplex of the outer kinetochore that is critical for proper chromosome segregation.
    [Show full text]
  • Microrna-301 Mediates Proliferation and Invasion in Human Breast Cancer
    Published OnlineFirst March 10, 2011; DOI: 10.1158/0008-5472.CAN-10-3369 Cancer Molecular and Cellular Pathobiology Research MicroRNA-301 Mediates Proliferation and Invasion in Human Breast Cancer Wei Shi1, Kate Gerster1, Nehad M. Alajez1,6, Jasmine Tsang1, Levi Waldron1, Melania Pintilie2, Angela B. Hui1, Jenna Sykes2, Christine P’ng4, Naomi Miller5, David McCready3, Anthony Fyles4, and Fei-Fei Liu1,4 Abstract Several microRNAs have been implicated in human breast cancer but none to date have been validated or utilized consistently in clinical management. MicroRNA-301 (miR-301) overexpression has been implicated as a negative prognostic indicator in lymph node negative (LNN) invasive ductal breast cancer, but its potential functional impact has not been determined. Here we report that in breast cancer cells, miR-301 attenuation decreased cell proliferation, clonogenicity, migration, invasion, tamoxifen resistance, tumor growth, and microvessel density, establishing an important oncogenic role for this gene. Algorithm-based and experimental strategies identified FOXF2, BBC3, PTEN, and COL2A1 as candidate miR-301 targets, all of which were verified as direct targets through luciferase reporter assays. We noted that miR-301 is located in an intron of the SKA2 gene which is responsible for kinetochore assembly, and both genes were found to be coexpressed in primary breast cancer samples. In summary, our findings define miR-301 as a crucial oncogene in human breast cancer that acts through multiple pathways and mechanisms to promote nodal or distant relapses. Cancer Res; 71(8); 2926–37. Ó2011 AACR. Introduction women presenting with lymph node negative (LNN) disease fare well (16); however, the development of metastatic relapse Since the initial discovery of microRNAs (miRNA) in 1993 is almost always incurable (17).
    [Show full text]
  • Single-Cell Level Spatial Gene Expression in the Embryonic Neural Differentiation Niche
    Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Single-Cell Level Spatial Gene Expression in the Embryonic Neural Differentiation Niche Yi Huang1,2, Xiaoming Yu1,3, Na Sun1, Nan Qiao1, Yaqiang Cao1,2, Jerome D. Boyd-Kirkup1, Qin Shen4,5 and Jing-Dong J. Han1# 1Chinese Academy of Sciences Key Laboratory of Computational Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031, China 2University of Chinese Academy of Sciences, Yuquan Road, Beijing, 100049, China. 3Center for Molecular Systems Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing, 100101, China 4Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, 100084, China 5Center for Stem Cell Biology and Regenerative Medicine, School of Medicine, Tsinghua University, Beijing, 100084, China # Correspondence should be addressed to J.-D.J.H (Tel.: 86-21-54920458 Email: [email protected]). 1 Downloaded from genome.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Running title: Single-Cell Level Gene Expression of Neurogenesis Keywords: single cell, neurogenesis, image analysis, chromatin remodeling, spatial gene expression. Abstract With the rapidly increasing availability of high-throughput in situ hybridization images, how to effectively analyze these images at high-resolution for global patterns and testable hypotheses has become an urgent challenge. Here we developed a semi-automated image analysis pipeline to analyze in situ hybridization images of E14.5 mouse embryos at single-cell resolution for over 1,600 telencephalon-expressed genes from the Eurexpress database.
    [Show full text]
  • Identification and Functional Analysis of SKA2 Interaction with The
    499 Identification and functional analysis of SKA2 interaction with the glucocorticoid receptor Lisa Rice1,2, Charlotte E Waters1, Jennifer Eccles1, Helen Garside1, Paula Sommer1, Paul Kay1, Fiona H Blackhall1, Leo Zeef2, Brian Telfer3, Ian Stratford3, Rob Clarke1, Dave Singh1, Adam Stevens1, Anne White1,2 and David W Ray1 1Faculty of Medical and Human Sciences, School of Medicine, 2Faculty of Life Sciences and 3Faculty of Medical and Human Sciences, School of Pharmacy, University of Manchester, Manchester M13 9PT, UK (Correspondence should be addressed to D Ray, Centre for Molecular Medicine, and Endocrine Sciences Research Group, Faculty of Medical and Human Sciences, University of Manchester, Stopford Building, Manchester M13 9PT, UK; Email: [email protected]) Abstract Glucocorticoid (GC) receptors (GRs) have profound anti- overexpression increased GC transactivation in HepG2 cells survival effects on human small cell lung cancer (SCLC). To while SKA2 knockdown in A549 human lung epithelial cells explore the basis of these effects, protein partners for GRs were decreased transactivation and prevented dexamethasone inhi- sought using a yeast two-hybrid screen. We discovered a novel bition of proliferation. GC treatment decreased SKA2 protein gene, FAM33A, subsequently identified as a SKA1 partner and levels in A549 cells, as did Staurosporine, phorbol ester and involved in mitosis, and so renamed Ska2. We produced an anti- trichostatin A; all agents that inhibit cell proliferation. Over- peptide antibody that specifically recognized full-length human expression of SKA2 potentiated the proliferative response to SKA2 to measure expression in human cell lines and tissues. IGF-I exposure, and knockdown with shRNA caused cells to There was a wide variation in expression across multiple cell lines, arrest in mitosis.
    [Show full text]
  • SKA2 (NM 001100595) Human Tagged ORF Clone 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 RC222296 SKA2 (NM_001100595) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: SKA2 (NM_001100595) Human Tagged ORF Clone Tag: Myc-DDK Symbol: SKA2 Synonyms: FAM33A Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin ORF Nucleotide >RC222296 representing NM_001100595 Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGGCCTCGGAGGTGGGGCACAATTTGGAGTCGCCGGAAACTCCGGGCGGCGGAGGCTGGACCAGAGTCG AGTTCCCTCCTCCTGCACCAAAGGGAGCCGCCACCGTCTGGTGTCTAAACCGCCTCGGTTCCAGAAAGCT GAGTCTGATCTGGATTACATTCAATACAGGCTGGAATATGAAATCAAGACTAATCATCCTGATTCAGCAA GTGAGCTGTCACCAC ACGCGTACGCGGCCGCTCGAGCAGAAACTCATCTCAGAAGAGGATCTGGCAGCAAATGATATCCTGGATT ACAAGGATGACGACGATAAGGTTTAA Protein Sequence: >RC222296 representing NM_001100595 Red=Cloning site Green=Tags(s) MASEVGHNLESPETPGGGGWTRVEFPPPAPKGAATVWCLNRLGSRKLSLIWITFNTGWNMKSRLIILIQQ VSCHH TRTRPLEQKLISEEDLAANDILDYKDDDDKV Chromatograms: https://cdn.origene.com/chromatograms/mk8041_a01.zip Restriction Sites: SgfI-MluI 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 SKA2 (NM_001100595) Human Tagged ORF Clone – RC222296
    [Show full text]