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Unformatted text preview: e indeed melanocytic cells. Immunofluorescent double‐staining was then used to further characterise the differentiation status of these cells. Based on the known PAX3‐regulated pathways involved in normal melanocyte development and differentiation, the following markers were used here to more precisely determine the differentiation status of the cells: HES1 (marker of less differentiated melanocytic cells), and MLANA (marker of differentiated melanocytes). Previous studies have implicated PAX3 in regulation of melanoma proliferation, survival and migration, providing us with an array of markers to use for further characterisation of PAX3‐expressing cells in melanoma tissue. Markers used were for: proliferation (Ki67), survival (BCL2L1), and migration (MCAM). Again co‐
expression of PAX3 with these markers was analysed by immunofluorescence. The frequency of PAX3 co‐expression with each marker was quantified in normal skin, in naevi, and in primary and metastatic melanoma samples. The results of these experiments were published in Medic and Ziman, 2010, the second publication towards this thesis (Chapter III). 29 Chapter I – Thesis Overview
Aim 3: To identify the roles of PAX3 by analysis of its target genes in melanocytes and in melanoma cells. To clarify the roles of PAX3 in melanocytes and melanoma cells we sought to identify its direct targets in both cells types. By investigating published literature we made a selection of confirmed and potential PAX3 target genes. These genes are representative of the specific PAX3‐dependant regulatory mechanisms involved in differentiation, proliferation, cell survival and migration. ChIP‐qPCR assay was used to analyse PAX3‐binding to the promoter of the selected genes in vitro, in a metastatic melanoma cell line, and in primary neonatal melanocytes. By investigating the difference/similarity in PAX3 binding to its targets between melanocytes and melanoma cells, we were able to identify mechanisms exclusive to melanoma cells, but also confirmed mechanisms of PAX3 regulation common to both melanocytes and melanoma cells. Levels of expression of target genes were quantified to substantiate th...
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- Spring '14