Fig. 3

NAT10 expression in PDAC regulates focal adhesion in an ac4C-dependent manner. (A) RNA dot blot of ac4C level in NAT10-KD PDAC cells. Methylene blue was used as a loading control. (B) HOMER motif analysis for ac4C sites with acRIP-seq (upper) and ac4C-seq (lower) in NAT10 KD and control PANC-1 cells. (C) Cumulative distribution showing differential expression of ac4C transcripts in NAT10 KD and control PANC-1 cells, based on acRIP-seq. Kolmogorov-Smirnov test (n = 3). (D) Frequency distribution of ac4C + transcript in PANC-1 cells from acRIP-seq. (E) Read distribution in ac4C-seq. n = 3. (F) Number of C to T single nucleotide variation in ac4C-seq. (G-H) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of acRIP-seq and ac4C-seq data indicates the enrichment of the focal adhesion pathway. (I) Immunoblot images of FAK, Src, phospho-FAK, phospho-Src, CDC42 and GAPDH, in NAT10 KD and control PDAC cells. (J) Immunoblotting image of NAT10, FAK, Src, phospho-FAK, phospho-Src and GAPDH, in NAT10 KD cells with overexpression of NAT10 wild-type (WT), overexpression of NAT10-G641E mutant and control PDAC cells. (K-L) Representative immunofluorescent staining (left) and mean fluorescence intensity (MFI) (right) of phosphor-Src (K) and phosphor-FAK (L) in PANC-1 cells. Scale bar: 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001. (K-L), One-way ANOVA