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Deciphering the Role of the Developmental Transcription Factor SOX6 in Tumorigenesis and Progression of Ewing Sarcoma

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Deciphering the Role of the Developmental Transcription Factor SOX6 in Tumorigenesis and Progression of Ewing Sarcoma Aus dem Pathologischen Institut der Ludwig-Maximilians-Universität München Direktor: Prof. Dr. med. Thomas Kirchner Deciphering the role of the developmental transcription factor SOX6 in tumorigenesis and progression of Ewing sarcoma Dissertation Zum Erwerb des Doctor of Philosophy (Ph.D) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München Vorgelegt von Aruna Marchetto aus München 2020 Mit Genehmigung der Medizinischen Fakultät der Universität München Supervisor(s): PD Dr. med. Thomas Grünewald, Ph.D. Prof. Dr. med. Thomas Kirchner Dean: Prof. Dr. med. dent. Reinhard Hickel Day of oral defense: 18.05.2020 Per i miei genitori Affidavit Pathologisches Institut der LMU Marchetto, Aruna Thalkirchner Str. 36 80337 München I hereby declare, that the submitted thesis entitled „Deciphering the roles of the developmental transcription factor SOX6 in tumorigenesis and progression of Ewing sarcoma“ is my own work. I have only used the sources indicated and have not made unauthorized use of services of a third party. Where the work of others has been quoted or reproduced, the source is always given. I further declare that the submitted thesis or parts thereof have not been presented as part of an examination degree to any other university. Munich, 20.05.2020 Aruna Marchetto Confirmation of congruency between printed and electronic version of the doctoral thesis Pathologisches Institut der LMU Marchetto, Aruna Thalkirchner Str. 36 80337 München I hereby declare that the electronic version of the submitted thesis, entitled „Deciphering the roles of the developmental transcription factor SOX6 in tumorigenesis and progression of Ewing sarcoma“ is congruent with the printed version both in content and format. Munich, 20.05.2020 Aruna Marchetto Table of contents Table of contents Summary ................................................................................................................................................. V 1. Introduction ............................................................................................................................. 1 1.1 Ewing Sarcoma ........................................................................................................................ 1 1.1.1 Origin ....................................................................................................................................... 1 1.1.2 Molecular and genetic profile of EwS ..................................................................................... 1 1.1.3 Clinical aspects: epidemiology, diagnosis and therapy ........................................................... 2 1.2 SRY-related HMG-box 6 (SOX6) ............................................................................................... 4 1.2.1 SOX family of transcription factors ......................................................................................... 4 1.2.2 SOX6 gene: Structure and regulation ...................................................................................... 5 1.2.3 SOX6 function in vertebrate development ............................................................................. 7 1.2.4 Role of SOX6 in endochondral ossification ............................................................................. 8 1.2.5 SOX6 expression in tumors and other malignancies ............................................................. 10 1.3 Oxidative stress and the role of TXNIP .................................................................................. 11 2 Research objectives and scientific aims ................................................................................ 13 2.2 Research objectives ............................................................................................................... 13 2.3 Scientific aims ........................................................................................................................ 13 3. Materials and methods ......................................................................................................... 14 3.1 Materials ................................................................................................................................ 14 3.1.1 List of manufacturers ............................................................................................................ 14 3.1.2 General materials .................................................................................................................. 15 3.1.3 Mice strains ........................................................................................................................... 16 3.1.4 Instruments and equipment .................................................................................................. 16 3.1.5 Chemicals ............................................................................................................................... 17 3.1.6 Biological reagents ................................................................................................................ 18 3.1.7 Commercial kits ..................................................................................................................... 19 3.1.8 Restriction enzymes .............................................................................................................. 20 3.1.9 Primary and Secondary antibodies for Western blot and immunohistochemistry .............. 20 3.1.9.1 Western blot .......................................................................................................................... 20 3.1.9.2 Immunohistochemistry ......................................................................................................... 20 3.1.10 Buffer and solutions .............................................................................................................. 20 3.1.11 SDS-PAGE gel compositions................................................................................................... 21 3.1.12 Sequences .............................................................................................................................. 21 3.1.12.1 shRNA sequences for pLKO-Tet-On cloning .......................................................................... 21 3.1.12.2 Sequences for pCDH vector cloning ...................................................................................... 22 Table of contents 3.1.12.3 Primer .................................................................................................................................... 22 3.1.12.4 Sequencing primer ................................................................................................................ 23 3.1.12.5 Sequences for small interfering RNAs (siRNA) ...................................................................... 23 3.1.13 Vectors ................................................................................................................................... 23 3.1.14 Software ................................................................................................................................ 23 3.2 Methods ................................................................................................................................ 24 3.2.1 Microbiology .......................................................................................................................... 24 3.2.1.1 Cloning with the pGL3-Promotor vector ............................................................................... 24 3.2.1.2 Cloning with the pLKO-Tet-On system .................................................................................. 28 3.2.1.3 Cumate-inducible pCDH-CuO-MCS-EF1αCymR-T2A-Puro ..................................................... 31 3.2.1.4 Transformation of E.coli and colony PCR .............................................................................. 33 3.2.1.5 Sequencing of pGL3-mSat, pLKO-shSOX6/shCtrl and pCDH-TXNIP constructs ..................... 36 3.2.2 Cell culture ............................................................................................................................. 37 3.2.2.1 Cell lines ................................................................................................................................. 37 3.2.2.2 Cell culture methods ............................................................................................................. 38 3.2.2.3 Transduction of pLKO-shSOX6 and pLKO-shSOX6-pCDH-TXNIP in EwS cell lines ................. 38 3.2.2.4 Transfection ........................................................................................................................... 39 3.2.3 Molecular biology .................................................................................................................. 40 3.2.3.1 Isolation of total RNA, cDNA synthesis and quantitative real time PCR (qRT-PCR) .............. 40 3.2.3.2 DNA extraction ...................................................................................................................... 41 3.2.4 Biochemistry .......................................................................................................................... 41 3.2.4.1 Generation of cell lysates and Bradford assay ...................................................................... 41 3.2.4.2 SDS-PAGE and Western Blot.................................................................................................. 42 3.2.5 Cell-based assays
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