DOCSLIB.ORG

SOX7 Is Down-Regulated in Lung Cancer

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SOX7 Is Down-Regulated in Lung Cancer Hayano et al. Journal of Experimental & Clinical Cancer Research 2013, 32:17 http://www.jeccr.com/content/32/1/17 RESEARCH Open Access SOX7 is down-regulated in lung cancer Takahide Hayano1, Manoj Garg1*, Dong Yin2, Makoto Sudo1, Norihiko Kawamata2, Shuo Shi3, Wenwen Chien1, Ling-wen Ding1, Geraldine Leong1, Seiichi Mori4, Dong Xie3, Patrick Tan1,5 and H Phillip Koeffler1,2,6 Abstract Background: SOX7 is a transcription factor belonging to the SOX family. Its role in lung cancer is unknown. Methods: In this study, whole genomic copy number analysis was performed on a series of non-small cell lung cancer (NSCLC) cell lines and samples from individuals with epidermal growth factor receptor (EGFR) mutations using a SNP-Chip platform. SOX7 was measured in NSCLC samples and cell lines, and forced expressed in one of these lines. Results: A notable surprise was that the numerous copy number (CN) changes observed in samples of Asian, non-smoking EGFR mutant NSCLC were nearly the same as those CN alterations seen in a large collection of NSCLC from The Cancer Genome Atlas which is presumably composed of predominantly Caucasians who often smoked. However, four regions had CN changes fairly unique to the Asian EGFR mutant group. We also examined CN changes in NSCLC lines. The SOX7 gene was homozygously deleted in one (HCC2935) of 10 NSCLC cell lines and heterozygously deleted in two other NSCLC lines. Expression of SOX7 was significantly downregulated in NSCLC cell lines (8/10, 80%) and a large collection of NSCLC samples compared to matched normal lung (57/62, 92%, p= 0.0006). Forced-expression of SOX7 in NSCLC cell lines markedly reduced their cell growth and enhanced their apoptosis. Conclusion: These data suggest that SOX7 is a novel tumor suppressor gene silenced in the majority of NSCLC samples. Keywords: CNAG, SNP-Chip, Lung cancer, SOX7, Promoter methylation Introduction myogenesis [8]. Recently, SOX7 has been proposed to Lung cancer is the leading cause of cancer-related function as a tumor suppressor in colorectal and death in the world. If surgery is inadequate, further prostate cancers [9,10]. We provide evidence that therapy is rarely curative. Understanding the genomic SOX7 behaves as a tumor suppressor in lung tissue abnormalities in this disease affords the opportunity and its expression is either low or silenced in the to identify new therapeutic targets. An example is the majority of lung cancers. use of Gefitinib for patients whose non-small cell lung cancer (NSCLC) has an epidermal growth factor receptor (EGFR) mutation in either exon 19 or 21. Materials and methods SOX7 is a member of the SOX (SRY-related high Cell lines and tissue samples mobility group box) transcription factors [1]. This Ten human lung cancer cell lines (H23, H460, protein, together with SOX17 and SOX18, comprises H820, H1299, H1975, HCC827, HCC2279, HCC2935, the SOX F subgroup [2] and helps mediate various HCC4006, PC14) were cultured in RPMI medium with developmental processes including a role in the 10% FBS and kept in a humidified atmosphere of regulation of hematopoiesis [3], cardiogenesis [4], 5% CO2. After IRB consent, total DNA and RNA of vasculogenesis [5,6], endoderm differentiation [7] and normal and cancerous lung tissues were obtained from the National University of Singapore (NUH-NUS * Correspondence: [email protected] Tissue Repository). Also, sixty-two pairs of primary 1Genomic Oncology Programme, Cancer Science Institute of Singapore, NUS, Singapore, 14 Medical Drive, #12-01, Singapore 117599, Singapore NSCLCs and their corresponding adjacent normal Full list of author information is available at the end of the article tissues, which were at least 5 cm away from the © 2013 Hayano 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. Hayano et al. Journal of Experimental & Clinical Cancer Research 2013, 32:17 Page 2 of 11 http://www.jeccr.com/content/32/1/17 Figure 1 Whole genome copy number analysis using high resolution SNP-Chips. (A, B) Heat map of DNA copy numbers found in the p and q arms of the chromosomes (horizontal axis) of 56 NSCLC samples from the TCGA data base [top panel, Figure 1A], 9 NSCLC patient samples with EGFR mutations [bottom panel, Figure 1A] and 8 NSCLC cell lines with EGFR mutations [Figure 1B] (vertical axis). DNA copy numbers are indicated by colors (black, blue, green, pink, orange and red are 0, 1, 2, 3, 4 and ≥5 copies, respectively). Common copy number gain regions are emphasized by red dotted rectangles. Common copy number loss region is emphasized by blue dotted rectangle. (C) At chromosome 8p23.1, a homozygous deletion of SOX7 occurs in the HCC2935 NSCLC cell line. Red dots show raw data. Blue line denotes total gene dosage by CNAG; level 2 indicates diploid (2N) amount of DNA. Sample is mostly hemizygous. Green small vertical bars immediately under the chromosome display heterozygous SNP sites. The bottom lines (Red and Green) denote allele-specific gene dosage (one line indicates gene dosage of the maternal allele, and the other indicates gene dosage of the paternal allele). Sample shows that chromosome 8 is hemizygously deleted except at 8p23.1 where the second allele is also lost in a small region resulting in homozygous deletion of the UNQ9391, RP1L1 and the SOX7 genes. Hayano et al. Journal of Experimental & Clinical Cancer Research 2013, 32:17 Page 3 of 11 http://www.jeccr.com/content/32/1/17 cancer, were obtained from NSCLC patients treated at Region (-687 to -440): 5’-TTAATTAGGTGGTTGAG Shanghai Chest Hospital (Shanghai, China), after their AATTAGAA and 5’-TAACCATAAACCCCTCAAAACA written informed consent. None of the patients Region (-71 to +251): 5’-TTTTGGAGAGTTATT received radio-chemotherapy prior to obtaining the GGAGGA and 5’-CCTTAACCCAAACCATAAAAA tissues. Lung cancer cells stably expressing either GFP PCR products were cloned into either the pGEM-T or or SOX7 were generated by transducing them with pGEM-T easy vector (Promega), and at least four clones PLKO.1 lentiviral vector system (Sigma). Briefly, cells from each sample were sequenced. were transduced with lentiviral vectors (SOX7 or GFP) at an MOI of 25 with 5 ug/ml polybrene added Methylation specific PCR (MSP) assay for 6 h. Twenty-four hours post-transduction, stable Primers specific for the unmethylated (U) and methyl- cells were selected using 1ug/ml puromycin for ated (M) sequences were designed by using Meth Primer 2-3 weeks. [13]. Primers sequences are as follows: MSP-U (-683 to -493): 5'-TAGGTGGTTGAGAATT High-density single nucleotide polymorphism-array analysis AGAATGAT G and 5'-CTTTCAAAAATAACCAAACT Genomic DNA from NSCLC cells were subjected to TCAAC GeneChip Human mapping (1000 K array for the EGFR MSP-M (-683 to 493): 5'-TTAGGTGGTTGAGAATT mutant lung cancer samples and 250 K array for the AGAACGAC and 5'-TCGAAAATAACCGAACTTCGA NSCLC cell lines). Both total and allelic-specific copy MSP-U (+192 to +321): 5'-ATAAGGGTTTTGAGAG numbers (CN) were determined using CNAG software TTGTATTTG and 5'-ACTCACCCAACATCTTACTA [11,12]. AACTCA MSP-M (+192 to +321): 5'-ATAAGGGTTTCGAGAG Quantitative real-time polymerase chain reaction TCGTATTC and 5'-TCACCCAACATCTTACTAAACT Real-time reverse transcriptase polymerase chain reaction CG (RT-PCR) was performed using Maxima® First Strand cDNA Synthesis Kit for RT-qPCR (Fermentas) according MTT assay to the manufacturer’s protocol. The expression level of H23 and H1975 cells were seeded at 5 × 103 per well in SOX7 mRNA in the samples was determined by quantita- 96-well plates. H1299 cells were seeded at 1.5 × 103 per tive real-time PCR (7500 Fast Real-Time PCR System, well in 96-well plates. MTT reagents were added to each Applied Biosystems) using KAPA™ SYBR® FAST qPCR Kit Master Mix (2X) Universal (Kapa Biosystems). Levels of Table 1 Common copy number genomic alterations in β-actin mRNA were used as an internal control. The delta NSCLC found in two cohorts: TCGA and EGFR mutant, threshold value (DCt) was calculated from the given non-smoking Asians threshold (Ct) value by the formula DCt = (Ct SOX7 – Ct Region of Chromosome Candidate target genes β-actin) for each sample. Gain of 1q21.1q-24.2 Large fragment Gain of 5p13.2 SKP2 Western blotting Gain of 7p11.2 EGFR NSCLC cells were lysed with ProteoJET™ Mammalian Gain of 8q24.3 PTP4A3 Cell Lysis Reagent (Fermentas). Immunoblotting was performed using either anti-SOX7 antibody (Sigma, Gain of 8q24.21 MYC, PVT1 HPA009065) or anti-β-actin antibody (Sigma, AC-15) Gain of 8q24.12 MTBP and either secondary anti-Rabbit IgG antibody (GE Loss of 8p23.1 UNQ9391, RP1|1, SOX7 Healthcare, NA934) or anti-murine IgG antibody (GE Gain of 11q13.2-13.3 CYCLIN D1, FGF3, FGF4, FGF19 β Healthcare, NA931), respectively. SOX7 or -actin bands Gain 12q14.2 TBK1, RASSF3 were detected using Pierce® Fast Western Blot Kit, Gain 12q14.3 HMGA2 SuperSignal® West Femto Substrate (Thermo SCIENTIFIC) and SuperSignal® West Pico Chemiluminescent Substrate Gain of 12q13.3-14.1 CDK4 (Thermo SCIENTIFIC), respectively. Gain of 12q12.1 KRAS Gain of 12q11.21 DDX11 Gain 14q13.3 PAX9 Bisulfite sequencing Gain of 17q12 Her2 Genomic DNA was modified by sodium bisulfite Gain of 17q25.3 TK1, BIRC5 using the CpGenome™ Turbo Bisulfite Modification Kit (MILLIPORE). The following PCR primers were used Common genomic alterations found in both NSCLC samples with EGFR mutations (9 samples) and those from the TCGA data base [56 samples, for bisulfite-modified genomic DNA [10]: probably these rarely have an EGFR mutation (Zhou et al. [14])]. Hayano et al.
Load more

Recommended publications
  • A Novel Tumor Suppressor Through the Wnt/Β-Catenin Signaling Pathway
    Liu et al. Journal of Ovarian Research 2014, 7:87 http://www.ovarianresearch.com/content/7/1/87 RESEARCH Open Access Reduced expression of SOX7 in ovarian cancer: a novel tumor suppressor through the Wnt/β-catenin signaling pathway Huidi Liu1,2,4, Zi-Qiao Yan1, Bailiang Li1, Si-Yuan Yin1, Qiang Sun1, Jun-Jie Kou1, Dan Ye1, Kelsey Ferns1,5, Hong-Yu Liu3 and Shu-Lin Liu1,2,4* Abstract Background: Products of the SOX gene family play important roles in the life process. One of the members, SOX7, is associated with the development of a variety of cancers as a tumor suppression factor, but its relevance with ovarian cancer was unclear. In this study, we investigated the involvement of SOX7 in the progression and prognosis of epithelial ovarian cancer (EOC) and the involved mechanisms. Methods: Expression profiles in two independent microarray data sets were analyzed for SOX7 between malignant and normal tissues. The expression levels of SOX7 in EOC, borderline ovarian tumors and normal ovarian tissues were measured by immunohistochemistry. We also measured levels of COX2 and cyclin-D1 to examine their possible involvement in the same signal transduction pathway as SOX7. Results: The expression of SOX7 was significantly reduced in ovarian cancer tissues compared with normal controls, strongly indicating that SOX7 might be a negative regulator in the Wnt/β-catenin pathway in ovarian cancer. By immunohistochemistry staining, the protein expression of SOX7 showed a consistent trend with that of the gene expression microarray analysis. By contrast, the protein expression level of COX2 and cyclin-D1 increased as the tumor malignancy progressed, suggesting that SOX7 may function through the Wnt/β-catenin signaling pathway as a tumor suppressor.
    [Show full text]
  • Regulation of Extra-Embryonic Endoderm Stem Cell Differentiation by Nodal and Cripto Signaling Marianna Kruithof-De Julio1,2, Mariano J
    DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 3885 Development 138, 3885-3895 (2011) doi:10.1242/dev.065656 © 2011. Published by The Company of Biologists Ltd Regulation of extra-embryonic endoderm stem cell differentiation by Nodal and Cripto signaling Marianna Kruithof-de Julio1,2, Mariano J. Alvarez2,3, Antonella Galli1,2, Jianhua Chu1,2, Sandy M. Price4, Andrea Califano2,3 and Michael M. Shen1,2,* SUMMARY The signaling pathway for Nodal, a ligand of the TGF superfamily, plays a central role in regulating the differentiation and/or maintenance of stem cell types that can be derived from the peri-implantation mouse embryo. Extra-embryonic endoderm stem (XEN) cells resemble the primitive endoderm of the blastocyst, which normally gives rise to the parietal and the visceral endoderm in vivo, but XEN cells do not contribute efficiently to the visceral endoderm in chimeric embryos. We have found that XEN cells treated with Nodal or Cripto (Tdgf1), an EGF-CFC co-receptor for Nodal, display upregulation of markers for visceral endoderm as well as anterior visceral endoderm (AVE), and can contribute to visceral endoderm and AVE in chimeric embryos. In culture, XEN cells do not express Cripto, but do express the related EGF-CFC co-receptor Cryptic (Cfc1), and require Cryptic for Nodal signaling. Notably, the response to Nodal is inhibited by the Alk4/Alk5/Alk7 inhibitor SB431542, but the response to Cripto is unaffected, suggesting that the activity of Cripto is at least partially independent of type I receptor kinase activity. Gene set enrichment analysis of genome-wide expression signatures generated from XEN cells under these treatment conditions confirmed the differing responses of Nodal- and Cripto-treated XEN cells to SB431542.
    [Show full text]
  • 8P23 Duplication Syndrome
    8p23 duplication syndrome rarechromo.org 8p23.1 duplication syndrome An 8p23.1 duplication is a very rare genetic condition in which there is a tiny extra piece from one of the 46 chromosomes – chromosome 8. Chromosomes are made up mostly of DNA and are the structures in the nucleus of the body’s cells that carry genetic information (known as genes), telling the body how to develop, grow and function. Chromosomes usually come in pairs: one chromosome from each parent. Of these 46 chromosomes, two are a pair of sex chromosomes: XX (a pair of X chromosomes) in females and XY (one X chromosome and one Y chromosome) in males. The remaining 44 chromosomes are grouped in 22 pairs, numbered 1 to 22 approximately from the largest to the smallest. Each chromosome has a short (p) arm (shown on the left in the diagram below) and a long (q) arm (on the right). Generally speaking, for correct development the right amount of genetic material is needed – not too little and not too much. However, a child’s other genes and personality also help to determine future development, needs and achievements. Looking at chromosome 8p23.1 You can’t see chromosomes with the naked eye, but if you stain them and magnify them under a microscope, you can see that each one has a distinctive pattern of light and dark bands (see diagram below). Band 8p23.1 contains around 6.5 million base pairs. This sounds like a lot, but it is actually quite small and is only 0.2 per cent of the DNA in each cell and only four per cent of the DNA on chromosome 8.
    [Show full text]
  • The Role of SOX7 in the Activation of Satellite Cells and Regulation of Skeletal Myogenesis
    The role of SOX7 in the activation of satellite cells and regulation of skeletal myogenesis. by Rashida Rajgara A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Master of Biochemistry In Department of Biochemistry, Microbiology and Immunology University of Ottawa Ottawa, ON, Canada © Rashida Rajgara, Ottawa, Canada, 2014. Abstract One of the major drawbacks of using stem cell therapy to treat muscular dystrophies is the challenge of isolating sufficient numbers of suitable precursor cells for transplantation. As such, a deeper understanding of the molecular mechanisms involved during muscle development, which would increase the proportion of embryonic stem cells that can differentiate into skeletal myocytes, is essential. In conditional SOX7-/- mice, we observed that the loss of SOX7 in satellite cells resulted in poor differentiation and fusion. In vivo, we observed fewer Pax7+ satellite cells in the mice lacking SOX7 as well as smaller muscle fibers. RT-qPCR data also revealed that Pax7, MRF and MHC3 transcript levels were down- regulated in SOX7 knockdown mice. Surprisingly, when SOX7 was overexpressed in embryonic stem cells, we found that there was a defect in making muscle precursor cells, specifically a failure to activate Pax7 expression. Taken together, these results suggest that SOX7 expression is required for the proper regulation of skeletal myogenesis. ii Acknowledgements I would love to take this opportunity to thank my supervisor Dr. Ilona Skerjanc for her trusting nature and always wanting to give a chance to every student, regardless. Her endless direction, support and scientific brainstorming sessions made my graduate student experience a lot easier.
    [Show full text]
  • The Transcription Factor Sox7 Modulates Endocardiac Cushion
    Hong et al. Cell Death and Disease (2021) 12:393 https://doi.org/10.1038/s41419-021-03658-z Cell Death & Disease ARTICLE Open Access The transcription factor Sox7 modulates endocardiac cushion formation contributed to atrioventricular septal defect through Wnt4/ Bmp2 signaling Nanchao Hong1,2, Erge Zhang1, Huilin Xie1,2,LihuiJin1,QiZhang3, Yanan Lu1,AlexF.Chen3,YongguoYu4, Bin Zhou 5,SunChen1,YuYu 1,3 and Kun Sun1 Abstract Cardiac septum malformations account for the largest proportion in congenital heart defects. The transcription factor Sox7 has critical functions in the vascular development and angiogenesis. It is unclear whether Sox7 also contributes to cardiac septation development. We identified a de novo 8p23.1 deletion with Sox7 haploinsufficiency in an atrioventricular septal defect (AVSD) patient using whole exome sequencing in 100 AVSD patients. Then, multiple Sox7 conditional loss-of-function mice models were generated to explore the role of Sox7 in atrioventricular cushion development. Sox7 deficiency mice embryos exhibited partial AVSD and impaired endothelial to mesenchymal transition (EndMT). Transcriptome analysis revealed BMP signaling pathway was significantly downregulated in Sox7 deficiency atrioventricular cushions. Mechanistically, Sox7 deficiency reduced the expressions of Bmp2 in atrioventricular canal myocardium and Wnt4 in endocardium, and Sox7 binds to Wnt4 and Bmp2 directly. Furthermore, 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; WNT4 or BMP2 protein could partially rescue the impaired EndMT process caused by Sox7 deficiency, and inhibition of BMP2 by Noggin could attenuate the effect of WNT4 protein. In summary, our findings identify Sox7 as a novel AVSD pathogenic candidate gene, and it can regulate the EndMT involved in atrioventricular cushion morphogenesis through Wnt4–Bmp2 signaling.
    [Show full text]
  • Download This PDF File
    The Ohio Journal of Volume 120 No. 1 April Program Abstracts SCIENCEOPEN ACCESS • ONLINE • INTERNATIONAL • MULTIDISCIPLINARY Special Notice The 129th Annual Meeting of The Ohio Academy of Science, described herein, was not held due to the coronavirus disease 2019 (COVID-19) pandemic. However, all abstracts were peer- reviewed and constitute valid scientific publications. The online version of this publication is available at https://doi.org/10.18061/ojs.v120i1.7574 . The Ohio Journal of SCIENCE ISSN: 0030-0950 (print) | ISSN: 2471-9390 (online) Open Access online EDITORIAL POLICY General The Ohio Journal of Science (OJS) has published peer-reviewed, Please contact the editor directly for general questions regarding original contributions to science, education, engineering, and content or appropriateness of submissions: technology since 1900. The OJS encourages submission of manuscripts relevant to Ohio, but readily considers all submissions Dr. Lynn E. Elfner, Editor—Email: [email protected] that advance the mission of The Ohio Academy of Science: To foster curiosity, discovery, innovation, and problem-solving skills in Ohio. Indexing The Academy produces two issues annually: peer-reviewed April The Ohio Journal of Science is indexed by: Program Abstracts (Issue No. 1) and peer-reviewed full papers Academic OneFile | Biological Abstracts/BIOSIS Previews in December (Issue No. 2). The Ohio State University Libraries CAB Abstracts | EBSCOhost Databases | Google Scholar publishes both issues Open Access online on behalf of The Ohio Knowledge Bank (The Ohio State University Libraries) Academy of Science. The Academy distributes a print version of ProQuest Databases | SciFinder Scholar | Zoological Record the April Program Abstracts at the annual meeting. Peer-reviewed articles are published as accepted throughout the year and compiled at year end into a single online volume.
    [Show full text]
  • A Neuro-Specific Hedgehog-Responsive Enhancer from Intron 1 of the Murine Laminin Alpha 1 Gene
    A neuro-specific hedgehog-responsive enhancer from intron 1 of the murine laminin alpha 1 gene Thesis presented for the degree of PhD by Kalin Dimitrov Narov Department of Biomedical Science University of Sheffield September 2013 i Acknowledgements I thank my supervisors Anne-Gaëlle Borycki and Philip Ingham for all expert guidance, patience, and for the opportunity to study vertebrate development in their laboratories. I also thank my advisors Pen Rashbass and Vincent Cunliffe for the helpful advices and their critical analyses on my work, and our collaborator Norris Ray Dunn for advising me on mouse transgenics and providing me with mouse embryos. Many thanks also to Shantisree Rayagiri, Joseph B. Pickering, Claire Anderson, Ashish Maurya, Weixin Niah, Harriet Jackson, Raymond Lee, Xingang Wang, Yogavali Pooblan for helping me with text formatting and embryo harvesting, and for providing me with reagents and advices. I am also grateful to the whole D-floor community, as well as to Martin Zeidler’s and Marcelo Rivolta’s labs for letting me use their equipment. Last but not least, I thank my family for the constant support and encouragement, and especially my parents for nurturing in me the love to nature and knowledge. Therefore, I dedicate this work to the memory of my father. ii Abstract Laminin alpha 1 (LAMA1) is a major component of the earliest basement membranes in the mammalian embryo. Disruption of the murine Lama1 gene result in lethal failure of germ layer differentiation and extraembryonic membrane formation at gastrulation stages, while conditional deletion of Lama1 leads to aberrant organization of retinal neurons and vasculature, and defects in cerebellar glia and granule cell precursors later in development.
    [Show full text]
  • Xenopus Early Primordial Germ Cell Development: Mining the PGC Transcriptome Amanda M
    © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev155978. doi:10.1242/dev.155978 RESEARCH ARTICLE A novel role for sox7 in Xenopus early primordial germ cell development: mining the PGC transcriptome Amanda M. Butler1, Dawn A. Owens1, Lingyu Wang2,* and Mary Lou King1,‡ ABSTRACT 1998). During cleavage stages, cells containing germ plasm undergo Xenopus primordial germ cells (PGCs) are determined by the asymmetric division so that the germ plasm is only inherited by one presence of maternally derived germ plasm. Germ plasm daughter cell termed the presumptive PGC (pPGC). Although components both protect PGCs from somatic differentiation and somatic determinants are partitioned into pPGCs during cleavage begin a unique gene expression program. Segregation of the stages, the genetic programs for somatic fate are not activated there germline from the endodermal lineage occurs during gastrulation, because of translational repression and transient suppression of RNA and PGCs subsequently initiate zygotic transcription. However, the polymerase II-regulated transcription (Lai and King, 2013; gene network(s) that operate to both preserve and promote germline Venkatarama et al., 2010). Segregation of the germline occurs at differentiation are poorly understood. Here, we utilized RNA- gastrulation when the germ plasm moves to a perinuclear location and sequencing analysis to comprehensively interrogate PGC and subsequent divisions result in both daughter cells, now termed PGCs, neighboring endoderm cell mRNAs after lineage segregation. We receiving germ plasm. PGCs then initiate their zygotic transcription identified 1865 transcripts enriched in PGCs compared with program driven by unknown maternal transcription factors. However, endoderm cells. We next compared the PGC-enriched transcripts the activated gene network necessary for proper PGC specification with previously identified maternal, vegetally enriched transcripts and and development has not been characterized in Xenopus.
    [Show full text]
  • Supplementary Material and Methods
    Supplementary Material and Methods Patients, controls and tissue handling Fresh frozen lymphoma biopsies were obtained from100 newly diagnosed cases of DLBCL. The diagnoses were based on standard histology and immunphenotyping according to the 2008 WHO lymphoma classification.1 The fraction of tumor cells was scored, and samples with more than 50% and 80% tumor cells were selected for DNA and RNA extraction, respectively. Peripheral blood B-lymphocytes (PBL-B) were obtained from random, anonymous donors and isolated using CD19+ selection kit on a RoboSep Device (Stemcell). Genomic DNA was isolated after proteinase K digestion using the Purescript DNA Isolation Kit (Gentra Systems). Paraffin- embedded tissue from the same patient with no morphological signs of DLBCL (“normal control tissue”) was used as control material. The allelic frequencies of the lymphoma associated sequence variants were analysed in 500 other alleles from the Danish population. Clinical data were obtained from the patient files and from the Danish lymphoma registry LYFO. All patients were treated with antracyclin containing regimens, however only 21 patients received immunotherapy with Rituximab. Approval of this study was obtained from the regional ethical committee. Cell lines Diffuse large B-cell derived lymphoma cell lines: Farage, DB1, HT, RL, and Toledo were purchased from the American Type Culture Collection (ATCC). The cells were cultured in RPMI 1640 medium with Glutamax supplemented with 10% fetal calf serum. Mutation detection 1 Detection of TET2 mutations The melting characteristics of each exonic region were calculated and appropriate GC- and AT- clamps were included in the PCR primers to modulate the melting properties into a two-domain profile.2 For TET2 sequences, PCR was performed in 15- L reactions containing 10 mM Tris- HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM cresol red, 12% sucrose, 10 pmol of each primer, 100 µM each dNTP, 10 ng of genomic DNA, and 0.8 units of hot-star Taq polymerase.
    [Show full text]
  • 8P23 Deletion Syndrome
    8p23 deletion syndrome rarechromo.org 8p23 deletion syndrome An 8p23 deletion means that the cells of the body have a small but variable amount of genetic material missing from one of their 46 chromosomes – chromosome 8. For healthy development, chromosomes should contain just the right amount of material – not too much and not too little. Like most other chromosome disorders, the incorrect amount of material increases the risk of birth defects, developmental delay and learning difficulties. However, the problems vary and depend very much on what genetic material is missing. Chromosomes are made up of DNA held together by proteins. They are rod-like structures in the nucleus of the body’s cells. They carry genetic information (known as genes) that tell the body how to develop, grow and function. Base pairs are the chemicals in DNA that form the ends of the ‘rungs’ of its ladder-like structure. Chromosomes usually come in pairs, with one chromosome of each pair inherited from the father and 1 base pair = bp the other from the mother. Of the 46 chromosomes, two 1,000 base pairs = 1kb are a pair of sex chromosomes, XX (a pair of X 1,000,000 base pairs = 1Mb chromosomes) in females and XY (one X chromosome and one Y chromosome) in males. The remaining 44 chromosomes are grouped in 22 pairs, numbered 1 to 22 approximately from the largest to the smallest. Each chromosome has a short (p) arm (shown at the top in the diagram on the next page) and a long (q) arm (the bottom part of the chromosome).
    [Show full text]
  • Sheet1 Page 1 Gene Symbol Gene Description Entrez Gene ID
    Sheet1 RefSeq ID ProbeSets Gene Symbol Gene Description Entrez Gene ID Sequence annotation Seed matches location(s) Ago-2 binding specific enrichment (replicate 1) Ago-2 binding specific enrichment (replicate 2) OE lysate log2 fold change (replicate 1) OE lysate log2 fold change (replicate 2) Probability Pulled down in Karginov? NM_005646 202813_at TARBP1 Homo sapiens TAR (HIV-1) RNA binding protein 1 (TARBP1), mRNA. 6894 TR(1..5130)CDS(1..4866) 4868..4874,5006..5013 3.73 2.53 -1.54 -0.44 1 Yes NM_001665 203175_at RHOG Homo sapiens ras homolog gene family, member G (rho G) (RHOG), mRNA. 391 TR(1..1332)CDS(159..734) 810..817,782..788,790..796,873..879 3.56 2.78 -1.62 -1 1 Yes NM_002742 205880_at PRKD1 Homo sapiens protein kinase D1 (PRKD1), mRNA. 5587 TR(1..3679)CDS(182..2920) 3538..3544,3202..3208 4.15 1.83 -2.55 -0.42 1 Yes NM_003068 213139_at SNAI2 Homo sapiens snail homolog 2 (Drosophila) (SNAI2), mRNA. 6591 TR(1..2101)CDS(165..971) 1410..1417,1814..1820,1610..1616 3.5 2.79 -1.38 -0.31 1 Yes NM_006270 212647_at RRAS Homo sapiens related RAS viral (r-ras) oncogene homolog (RRAS), mRNA. 6237 TR(1..1013)CDS(46..702) 871..877 3.82 2.27 -1.54 -0.55 1 Yes NM_025188 219923_at,242056_at TRIM45 Homo sapiens tripartite motif-containing 45 (TRIM45), mRNA. 80263 TR(1..3584)CDS(589..2331) 3408..3414,2437..2444,3425..3431,2781..2787 3.87 1.89 -0.62 -0.09 1 Yes NM_024684 221600_s_at,221599_at C11orf67 Homo sapiens chromosome 11 open reading frame 67 (C11orf67), mRNA.
    [Show full text]
  • OCT4 Induces Embryonic Pluripotency Via STAT3 Signaling and Metabolic Mechanisms
    OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms Giuliano G. Stirparoa,b,1,2, Agata Kurowskia,c,1, Ayaka Yanagidaa,b, Lawrence E. Batesa,d, Stanley E. Strawbridgea, Siarhei Hladkoua,d, Hannah T. Stuarta, Thorsten E. Boroviake,f, Jose C. R. Silvaa,d, and Jennifer Nicholsa,e,f,2 aWellcome Trust—Medical Research Council Stem Cell Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, CB2 0AW Cambridge, United Kingdom; bLiving Systems Institute, University of Exeter, EX4 4QD Exeter, United Kingdom; cDepartment of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029; dDepartment of Biochemistry, University of Cambridge, CB2 1GA Cambridge, United Kingdom; eDepartment of Physiology, Development and Neuroscience, University of Cambridge, CB2 3EG Cambridge, United Kingdom; and fCentre for Trophoblast Research, University of Cambridge, CB2 3EG Cambridge, United Kingdom Edited by Rudolf Jaenisch, Whitehead Institute for Biomedical Research, Cambridge, MA, and approved December 8, 2020 (received for review June 3, 2020) OCT4 is a fundamental component of the molecular circuitry key pluripotency network gene as a contributing feature of the governing pluripotency in vivo and in vitro. To determine how OCT4 null phenotype. To date, evidence that all cells in OCT4 null OCT4 establishes and protects the pluripotent lineage in the embryos adopt a TE identity is largely restricted to morphology and embryo, we used comparative single-cell transcriptomics and quan- expression of TE-specific markers at the time of implantation (13, titative immunofluorescence on control and OCT4 null blastocyst 24, 26). To scrutinize how acquisition of pluripotency fails in inner cell masses at two developmental stages.
    [Show full text]