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. 2023 Mar 11;42(17):1360–1373. doi: 10.1038/s41388-023-02631-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — A Immunohistochemical staining of EZH2 and UBE2L6 in representative Schmorl’s stained pigmented human melanomas. Scale bar: 50 μm. B UBE2L6 protein scores (x-axis) negatively correlated with EZH2 protein scores (y-axis) in patient melanomas. Pink dots correspond to non-pigmented and purple dots to pigmented patient samples. p-value calculated from a linear regression analysis. R = correlation coefficient. Protein score = the percentage of immunopositive cells × immunostaining intensity. C–F Indicated cell lines were treated with 2 µM 5’-Azacitidine or DMSO (vehicle) for 72 h prior to: (C) methylation specific primer (MSP) analysis of methylation at the UBE2L6 promoter (M: Methylated specific primer; U: Unmethylated specific primer; number below image represents percent ratio of methylated (m) to unmethylated (u) DNA quantified by ImageJ after normalization to controls (m/u), (D) UBE2L6 mRNA quantification with RT-qPCR, (E) cell number by Trypan blue cell counting, and (F) EZH2 protein quantification with western blot. G Correlation between UBE2L6 and UHRF1 mRNA levels in TCGA cutaneous melanoma samples. H Kaplan-Meier curves of overall survival of TCGA cutaneous melanoma patients (n = 427) stratified by UHRF1 mRNA levels. p = 0.015 (log-rank test). I–K A375 human melanoma cells were transfected with one of two siRNAs against Uhrf1, or scrambled controls, followed by: (I) MSP analysis of UBE2L6 promoter as in (C), (J) EZH2 and UBE2L6 protein estimation by western blot, and (K) cell number by Trypan blue cell counting. L Immunohistochemical staining of UHRF1 and UBE2L6 in representative Schmorl’s stained pigmented human melanomas. Scale bar, 50 μm. M UBE2L6 protein scores (x-axis) in melanoma samples negatively correlated with UHRF1 scores (y-axis) in individual patients. Pink dots correspond to non-pigmented and purple dots to pigmented patient samples. P-value calculated from linear regression analysis. R = correlation coefficient. Protein score = percentage of immunopositive cells × immunostaining intensity. Data for D, E and K from three independent experiments are presented as means ± SD, analyzed by one-way ANOVA plus Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.