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. 2021 May 28;49(10):5779–5797. doi: 10.1093/nar/gkab415

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© The Author(s) 2021. 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 (http://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 5. — Global gene expression profiling indicates ALKBH5 downstream targets related to DNA damage repair are induced by ROS. (A, B) The frequency distribution of m⁶A peaks across the length of mRNA transcripts shown by metagene in control cells (A) or in cells expressing ALKBH5 K86/321R (B) with or without H₂O₂ treatment. Each region of the 5′ untranslated region (5′ UTR), coding region (CDS), and 3′ untranslated region (3′ UTR) was split into 100 segments, and the percentage of m⁶A peaks that fall within each segment was determined. (C, D) The density (line) and frequency (histogram) of m⁶A peaks in control samples (C) or SD-mutant overexpressed samples (D) with or without H₂O₂ treatment. (E) The adjusted density (line, top) and distribution (histogram, bottom) of hyper peaks from (C) across different mRNA regions in control samples with or without H₂O₂ treatment. (F) Enrichment analysis for significantly increased peaks of m⁶A modification from (C) in control samples with or without H₂O₂ treatment. (G) Differentially expressed genes shown in volcano figure in control samples with or without H₂O₂ treatment. There were 1051 genes withy significantly reduced expression (log₂FC < 0, P < 0.01), 1106 genes with significantly increased expression (log₂FC > 0, P < 0.01) and 15 102 genes without statistically significant changes in expression. (H) Differentially expressed genes shown in volcano figure in SD-mutant ALKBH5 overexpressing cells with or without H₂O₂ treatment. Almost all the transcripts show negligible expression changes. (I) m⁶A peak visualization of key transcripts in DNA repair in control samples with or without H₂O₂ treatment. (J) qRT-PCR analyses showing that ROS markedly up-regulates transcription of the three selected target genes (FAAP20, ATRX and RRC2). (K) mRNA m⁶A methylation validation of the three selected target genes (FAAP20, ATRX and RRC2) by MeRIP analysis. (L) ALKBH5 RIP analyses showing that H₂O₂-induecd ROS dramatically decreases ALKBH5 enrichment at FAAP20, ATRX and RFC2 mRNAs. (M–O) mRNAs half-life of the three selected target genes (FAAP20, ATRX and RRC2), with or without ROS treatment.