Figure 6. SHARPIN regulation of PRMT5 activity controls SOX10 expression and growth in MTAP-deleted human melanomas.

(A) Expression of MTAP and CDKN2A genes in melanoma patients (TCGA, n = 472). Green and red vertical lines indicate the cutoffs for classification as low-MTAP (n = 70) and high-MTAP (n = 50) melanomas, respectively. The green horizontal line indicates the expression level in normal tissue (TCGA, n = 1). (B) Pearson’s correlation coefficients for SHARPIN and SOX10 or SHARPIN and PAX3 expression in all (n = 472, blue), MTAP-low (n = 70, green), and MTAP-high (n = 50, red) melanoma samples. (C) Immunoblot analysis of the indicated proteins in melanoma cells with high, medium, or low sensitivity to growth inhibition by SHARPIN KD. (D) Immunoblot analysis (left upper) and colony-forming efficiency (lower) assay of WM35 cells expressing empty vector (control) or overexpressing SHARPIN. Cells were treated with vehicle (DMSO) or MTA (100 μM) for 72 hours. For CFE, cells were seeded at 10³/well and colonies were visualized and quantified (lower right) after 14 days in culture. PRMT5 activity (upper right) was assessed in anti-PRMT5–immunoprecipitated cell lysates. The quantitation data are presented as mean ± SD. n = 3 (lower right). n = 6 (upper right). Statistical significance was calculated using 2-way ANOVA (Tukey’s test). *P < 0.05; ***P < 0.0005. (C and D) Data shown represent at least 2 independent experiments.