Fig. 4

ITGB5 is the downstream target of ac4C modification in PDAC cells. (A-B) Gene-set enrichment analysis (GSEA) from GSE16515 (A) and our RNA-seq data (B) revealed that the focal adhesion pathway and related cell adhesion molecules were enriched in high NAT10 group. (C) Gene ontology (GO) analysis of our RNA-seq data showing enrichment of NAT10 KD cells, indicating that downregulated genes are related to cell adhesion mediated by integrin. (D) Heap map of focal adhesion pathways genes from RNA-seq analysis. Z-score was used. (E) Venn diagram of overlapping enriched genes of the focal adhesion pathway (KEGG) from acRIP-seq and ac4C-seq data. (E) Venn diagram of overlapping genes from acRIP-seq, RNA-seq, and ac4C-seq showing the downregulated genes in NAT10 knockdown PANC-1 cells. (F-H) Relative NAT10 and ITGB5 mRNA (F-G) and protein (H) expression in NAT10 KD and control PDAC cells. n = 3. (I) CPTAC database showing the correlation between NAT10 and ITGB5 mRNA levels. (J) Integrative Genomics Viewer (IGV) browser showing the ac4C peaks in ITGB5 CDS region. (K) ac4C RNA-immunoprecipitation (acRIP) followed by qRT-PCR analysis showing the downregulated ac4C level of ITGB5 in NAT10 KD cells. (L-M) Relative ITGB5 mRNA (K) and NAT10 and ITGB5 protein (L) expression in NAT10 KD PDAC cells with overexpression of the NAT10-WT and NAT10-G641E mutant. (N) acRIP-PCR analysis indicating the ac4C positive level of ITGB5 mRNA in NAT10 KD treated with overexpression of NAT10 wild-type (WT), overexpression of NAT10-G641E mutant, and control PANC-1 (left) and MIA PaCa-2 (right) cells. *p < 0.05, **p < 0.01, ***p < 0.001. (G), (L), and (N), One-way ANOVA. (K), Student’s test