SOX10 knockdown results in elevated SOX9 expression in mouse and human melanocytes.

<p><b>A</b>, Experimental design used to investigate the level of SOX9 and SOX10 expression <i>in vitro</i>. Cultured human keratinocytes, melanocytes, cells derived from biopsies of patients with giant congential naevi and melanoma cells (M010817 cell line) were subjected to RNA isolation and subsequent Q-RT-PCR analysis. Keratinocytes were used as a control. <b>B, C</b>, Quantitative real-time PCR analysis showing the decline of SOX9 expression (<b>C</b>) and increase of SOX10 expression (<b>B</b>) that correlate with the acquisition of malignant state by human NRAS^Q61K-mutated cells. Data are presented as the mean fold change and are normalized over levels found in melanocytes. <b>D, E</b>, SOX10 and SOX9 expression in a large set of proliferative and invasive cell lines analysed by gene expression using microarrays (<b>D</b>) and Western blot (<b>E</b>) techniques. <b>F</b>, Experimental design used to deregulate SOX10 expression in human cells derived from the biopsy of a patient with NRAS^Q61K-mutated giant congenital naevus. <b>G, H</b>, Quantitative real-time PCR analysis of SOX10 (<b>G</b>) and SOX9 (<b>H</b>) expression after the knockdown of SOX10. <b>I</b>, Experimental design used to analyze the expression of Sox9 in the melanocytic lineage from <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61K</i> <i>Sox10^LacZ/+</i> mice. <b>K, L</b>, Cells were isolated from the trunk skin of <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61K</i> <i>Sox10^LacZ/+</i> mice and stained for Melan-a and c-Kit antibodies. FACS-sorted cells were subsequently used for the RNA isolation and quantitative real-time PCR with primers specific for the coding regions of <i>Sox9</i> gene. Data are presented as the mean fold change and are normalized to the control. Kerat, keratinocytes; M, melanocytes; Nev, naevus cells; Mel, melanoma cells; KD, knock down.