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Expression, Functions, and New Target Genes of the Transcription Factor SOX10 in Human Melanoma

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Expression, Functions, and New Target Genes of the Transcription Factor SOX10 in Human Melanoma Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Expression, functions, and new target genes of the transcription factor SOX10 in human melanoma Saskia Anna Graf aus Neumarkt in der Oberpfalz 2016 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Frau Prof. Dr. Carola Berking betreut und von Herrn Prof. Dr. Karl-Peter Hopfner von der Fakultät für Chemie und Pharmazie vertreten. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 01.03.2016 …………………………… Saskia Graf Dissertation eingereicht am 04.03.2016 1. Gutachter: Prof. Dr. Karl-Peter Hopfner 2. Gutachterin: Prof. Dr. Carola Berking Mündliche Prüfung am 20.05.2016 “The storms come and go, the waves crash overhead, the big fish eat the little fish, and I keep on paddling.” (Lord Varys) - George R. R. Martin, A Clash of Kings Diese Arbeit widme ich meinen Eltern und meinem Mann Simon This thesis has been prepared in the laboratory of Prof. Dr. Carola Berking at the Department of Dermatology of the Ludwig Maximilian University of Munich. Parts of this thesis were published in the Journal of Investigative Dermatology in 2014: Graf SA, Busch C, Bosserhoff AK, Besch R, Berking C. SOX10 promotes melanoma cell invasion by regulating melanoma inhibitory activity. Journal of Investigative Dermatology 134: 2212-20 (2014) A manuscript containing other parts of this thesis is currently in preparation for submission to the Journal of Investigative Dermatology: Graf SA, Krebs S, Hornig E, Heppt MV, Kammerbauer C, Hamel A, Besch R, Blum H, Berking C. The myelin protein PMP2 is regulated by SOX10 and drives melanoma cell invasion. Tabel of contents Table of contents 1 Summary ............................................................................................................................. 1 Zusammenfassung ..................................................................................................................... 3 2 Introduction ......................................................................................................................... 5 2.1 Melanocyte Development ............................................................................................. 5 2.2 From melanocytes to melanoma .................................................................................. 8 2.3 Malignant melanoma .................................................................................................. 11 2.4 Melanoma therapy and resistance .............................................................................. 12 2.5 Linking melanomagenesis to the embryonic development of melanocytes ................. 15 2.6 SOX transcription factors in development and disease ............................................... 17 2.7 Aims of this study ....................................................................................................... 21 3 Materials and Methods ...................................................................................................... 22 3.1 Materials .................................................................................................................... 22 3.1.1 Media .................................................................................................................. 22 3.1.2 Buffers and solutions ........................................................................................... 23 3.1.2.1 Buffers and solutions for immunoblotting ...................................................... 24 3.1.2.2 Buffers and solutions for fluorescence-activated cell sorting ........................ 25 3.1.2.3 Buffers and solutions for luciferase reporter assay ....................................... 26 3.1.2.4 Buffers and solutions for electrophoretic mobility shift assay ........................ 26 3.1.2.5 Buffer for immunohistochemistry .................................................................. 26 3.1.2.6 Buffers for chromatin immunoprecipitation ................................................... 27 3.1.2.7 Further buffers and solutions ........................................................................ 28 3.1.3 Commercial kits ................................................................................................... 28 3.1.4 Transfection reagents.......................................................................................... 30 3.1.5 Oligonucleotides .................................................................................................. 30 3.1.5.1 Primers for quantitative real-time PCR ......................................................... 30 3.1.5.2 Primers for polymerase chain reaction ......................................................... 32 3.1.5.3 Oligonucleotides for electrophoretic mobility shift assay ............................... 34 3.1.5.4 Small interfering ribonucleic acids ................................................................ 35 3.1.6 Plasmids and vectors .......................................................................................... 35 3.1.7 Enzymes and polypeptides ................................................................................. 37 3.1.8 Antibodies ........................................................................................................... 38 3.1.8.1 Primary antibodies ....................................................................................... 38 IV Tabel of contents 3.1.8.2 Secondary antibodies ................................................................................... 39 3.1.9 Cell lines ............................................................................................................. 39 3.1.10 Appliances .......................................................................................................... 40 3.1.11 Consumables ...................................................................................................... 41 3.1.12 Software .............................................................................................................. 42 3.2 Methods ..................................................................................................................... 44 3.2.1 Cell culture .......................................................................................................... 44 3.2.1.1 Cultivation of human melanoma cell lines..................................................... 44 3.2.1.2 Isolation of melanoma cells from patient samples ........................................ 44 3.2.1.3 Cultivation of primary skin cells .................................................................... 45 3.2.2 Molecular biological methods .............................................................................. 45 3.2.2.1 Gene silencing using RNA interference ........................................................ 45 3.2.2.1.1 Design of small interfering RNAs ................................................................ 45 3.2.2.1.2 Transfection of siRNAs ............................................................................... 45 3.2.2.2 Transient and stable transfection of plasmid DNA ........................................ 46 3.2.2.3 Cell invasion assays ..................................................................................... 46 3.2.2.3.1 Matrigel invasion assay .............................................................................. 46 3.2.2.3.2 Spheroid assay .......................................................................................... 47 3.2.2.3.3 Chick embryo invasion assay ..................................................................... 48 3.2.2.4 Cell viability assay ........................................................................................ 49 3.2.2.5 Flow cytometry ............................................................................................. 50 3.2.2.5.1 Cell cycle analysis ...................................................................................... 50 3.2.2.5.2 Cell death analysis ..................................................................................... 50 3.2.2.6 Luciferase reporter assay ............................................................................. 50 3.2.2.7 Cloning of expression vectors ...................................................................... 51 3.2.2.8 Transformation and conservation of chemical competent Escherichia coli ... 52 3.2.3 Biochemical methods .......................................................................................... 53 3.2.3.1 Isolation of nucleic acids .............................................................................. 53 3.2.3.1.1 Plasmid isolation ........................................................................................ 53 3.2.3.1.2 RNA isolation ............................................................................................. 53 3.2.3.2 Quantification of gene expression ................................................................ 54 3.2.3.2.1 Copy DNA synthesis .................................................................................. 54 3.2.3.2.2 Quantitative real-time PCR ......................................................................... 54 V Tabel of contents 3.2.3.3 Copy DNA sequencing ................................................................................. 56 3.2.3.4 RNA sequencing .........................................................................................
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