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. 2023 Dec 20;52(5):2273–2289. doi: 10.1093/nar/gkad1193

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Histone lactylation potentiates the expression of ALKBH3. (A) Correlation analysis of ALKBH3 expression and LDHA or LDHB expression in the TCGA-UM cohort (n = 80). Significance was determined by Pearson correlation analysis (LDHA: R = 0.4, P = 0.00023, LDHB: R = 0.46, P = 0.000018). (B) Immunofluorescence of ALKBH3 (green), Pan Kla (red) and DAPI (blue) in tumor and normal samples. Scale bars: left panel, 100 μm; right panel, 20 μm. Correlation analysis of the relative protein expression of ALKBH3 and Pan Kla level in ocular melanoma cells and normal melanocytes. Significance was determined by Pearson correlation analysis (R= 0.5515, P < 0.0001). (C) Immunofluorescence of ALKBH3 (green), H3K18la (red) and DAPI (blue) in tumor and normal samples. Scale bars: left panel, 100 μm; right panel, 20 μm. Correlation analysis of the relative protein expression of ALKBH3 and H3K18la level in ocular melanoma cells and normal melanocytes. Significance was determined by Pearson correlation analysis (R= 0.6214, P < 0.0001). (D) IGV tracks from ChIP-seq analysis showing H3K18la enrichment at the promoter of ALKBH3. Sites a–d are distributed in the ALKBH3 genomic region, and sites b and c are the H3K18la peaks. Biological duplicates were analyzed. (E) IGV tracks from CUT&Tag analysis showing H3K18la enrichment at the promoter of ALKBH3. Sites a–d are distributed in the ALKBH3 genomic region, and sites b and c are the H3K18la peaks. Biological duplicates were analyzed. (F) ChIP-qPCR assay of H3K18la status in the ALKBH3genomic region in ocular melanoma cells (92.1 and CRMM1) upon treatment with histone lactylation inhibitors (2-DG or oxamate) or LDHA/B inhibition. The data are presented as the mean ± SD of experimental triplicates. (G) qPCR data showing ALKBH3 expression in ocular melanoma cells (92.1 and CRMM1) upon treatment with histone lactylation inhibitors (2-DG or oxamate) or LDHA/B inhibition. The data are presented as the mean ± SD of experimental triplicates. Significance was determined by unpaired two-tailed Student's t test. **P < 0.01, ***P < 0.001, ****P < 0.0001. (H and I) Western blot showing ALKBH3 expression relative to ACTB expression and Pan Kla, H3K18la expression relative to Histone H3 expression in ocular melanoma cells (92.1 and CRMM1) upon histone lactylation inhibitors (2-DG or oxamate). (J) Western blot showing LDHA, LDHB, ALKBH3 expression relative to ACTB expression and Pan Kla, H3K18la expression relative to Histone H3 expression in ocular melanoma cells (92.1 and CRMM1) upon LDHA/B inhibition. Nala, sodium lactate.