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. 2023 Sep 15;13(9):e1389. doi: 10.1002/ctm2.1389

FIGURE 4.

FIGURE 4

Neutrophil extracellular traps (NETs) promote METTL3 transcription via activation of H3K27ac signalling, catalysed by p300. (A and D) The level of m6A modification was analysed by dot blot assay. (B) RNA sequencing (RNA‐seq) identified the upregulation of METTL3 in NET‐treated alveolar epithelial cells. (C) Real‐time quantitative PCR (RT‐qPCR) confirmed the upregulation of METTL3 and IGF2BP2. (E and F) Western blot analysis shows that NETs increased p300, METTL3 and H3K27ac. While DNase I reversed these changes, (G) p300 inhibitor C646 reduced protein levels of H3K27ac and METTL3 in Western blot analysis. (H) C646, confirmed by RT‐qPCR, inhibited the relative METTL3 mRNA level. (I–K) Based on the Western blot and RT‐qPCR results, the protein and mRNA levels of METTL3 decreased due to the silencing of p300. (L) The chromatin immunoprecipitation (ChIP) results confirmed that the knockdown of p300 decreased the enrichment of H3K27ac signalling. ** p < .01 (two‐way analysis of variance [ANOVA] with Tukey's correction).