Fig. 3

NAT10 maintains the stability of MDM2 mRNA via ac4C modification. A Heat map showing differentially expressed genes identified by RNA-seq in NAT10-knockout cells relative to the control cells. Green and red indicate low and high mRNA levels, respectively. B Volcano plot of altered ac4C peaks within mRNA transcripts between NAT10-knockout and control cells. C A Venn diagram shows overlapping mRNA transcripts that were both differentially expressed (DEG) and hypoacetylated upon NAT10 knockout and differentially expressed genes (DEG) following NAT10 knockdown. D The ac4C peak was enriched in the 3′UTR of MDM2 from the acRIP-seq data. Squares indicate a significant decrease in the ac4C peak in NAT10-knockout cells relative to control AGS cells. E The relative ac4C levels of the MDM2 transcript were evaluated in the indicated cells by acRIP-qPCR. F RIP assay with anti-NAT10 and anti-IgG antibodies was carried out to analyze the relative NAT10 enrichment in MDM2 mRNA. G The mRNA levels of MDM2 in the indicated cells. H MDM2 mRNA levels were measured in cells treated with Remodelin for 24 h. I and J MDM2, p53, p21 and PUMA were detected by Western blotting in the indicated cells. K Western blot analysis of NAT10, MDM2 and p53 in xenograft tumor tissues. L MDM2 mRNA stability assessment in the indicated cells treated with 5 μg/mL actinomycin D (ACD). M A schematic diagram illustrating the luciferase reporter plasmids containing the wild-type (Wt) MDM2 3’UTR fragment or its mutant (Mut) counterpart that lacks the ac4C peak region. N and O The relative mRNA expression (N) and activity (O) of firefly luciferase fused with the wild-type or mutant MDM2 3′UTR in control AGS cells, NAT10-knockout cells and knockout cells re-expressing wild-type or mutant NAT10. P Immunoblot of p53 protein and quantification of the relative level of p53 at the indicated time in control and NAT10-knockout AGS cells after treatment with 100 μg/ml cycloheximide (CHX) to block protein synthesis. Error bars represent the SD from three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001. ns, not significant. All P values were determined by two-tailed t-test