Unformatted text preview: HEM1455 primary melanocytes and A2058 melanoma cells We have previously reported persistent PAX3 expression in melanocytic lesions and normal skin melanocytes, both follicular and epidermal . Here we confirm PAX3 expression in both HEM1455 and A2058 cell lines (Fig. 4.1), showing slightly lower (however not statistically significant) PAX3 levels in A2058 compared to HEM1455 cells. This is most likely due to the variable expression of PAX3 in A2058 cells, as evidenced by differences in PAX3‐staining intensity across the cultured cells, including some PAX3‐negative cells. Others have also reported high variation in PAX3 levels across a variety of melanoma cell lines, but more uniform staining and higher expression in melanocytes . Figure 4.1. PAX3 expression in melanocytes and melanoma cells. (A) PAX3 expression in A2058 cells confirmed by immunocytochemistry, showing co‐expression of PAX3 with MITF, a known marker of the melanocytic lineage. (B) PAX3 expression in HEM1455 and A2058 cells confirmed by western blotting. (C) Graph shows PAX3 mRNA levels in HEM1455 and A2058 (relative to GAPDH) assessed by RT‐qPCR. 83 Chapter IV – Medic, Rizos and Ziman, 2011 PAX3 target genes in HEM1455 and A2058 cell lines In order to assess PAX3 function/s and identify differential regulation mediators between melanocytes and melanoma cells, we have analysed 12 out of 56 potential direct PAX3 target genes (Supplementary Table S3). These genes were selected as indicators of key PAX3‐regulated processes, ie differentiation (SOX9, NES, and Hairy and enhancer of split 1 (HES1)), proliferation (TPD52, Cyclin A2 (CCNA2), and Nuclear factor kappa β 2 (NFKβ2)), cell survival (BCL2L1, PTEN, and Transforming growth factor‐beta 1 (TGFβ1) and migration (MCAM, CSPG4, and CXC chemokine receptor 4 (CXCR4)). In addition MITF and DCT, known PAX3 targets in the melanocytic cell lineage, were chosen as positive controls to assess ChIP assay efficiency. ChIP with anti‐
PAX3 antibody (DSHB) showed enrichment for both MITF and DCT promo...
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This document was uploaded on 03/06/2014 for the course CHEMISTRY 12 at National University of Singapore.
- Spring '14