Fig. 3. The NAT2-specific substrate APA causes mitotic arrest in slow NAT2 cells.

a NAT2 mediates APA acetylation. The velocity of NAPA formation was determined by LC-MS/MS in slow (green), rapid (yellow) NAT2 clones, vector control (blue), and parental RKO cells (black) after incubation with APA at indicated concentrations. b APA is selectively metabolized by NAT2. Enzyme kinetics of NAPA formation by human recombinant NAT1 (black) and NAT2 (red) proteins. c, d siRNA-mediated gene silencing of protein kinases with differential inhibition by APA and NAPA expressed in RKO CRC cells. Relative growth of RKO (c) and DLD-1 (d) cells transfected with a pool of siRNA targeting DYRK1A, AURKA, CDK7, non-target RNA control and mock transfection as percentage of the growth of untransfected cells. Mean and S.D. (error bars) of three independent experiments is shown. Data were analyzed by one-way ANOVA. *P = 0.0435, **P = 0.0013, ***P = 0.0004. e APA (2 µM) reduces the phospho-Thr288 Aurora kinase A (pAURKA) staining in NAT2-deficient cells but not in rapid NAT2 clones. Mitotic spindles were visualized by α-tubulin staining (green) and pAURKA levels (red). Representative images of three independent repeats are shown for each cell type. Scale bar, 10 µm. f Quantification of pAURKA signal in parental DLD-1 and slow and rapid NAT2 clones after APA treatment (2 µM). The pAURKA levels are shown as a fold of the DMSO control group. Mean and S.D. (error bars) of three independent experiments. Data were analyzed using two-way ANOVA. n.s., P = 0.0619 and ****P < 0.0001. g APA (2 µM), similarly to the AURKA selective inhibitor S1451 (10 µM) reduces the total protein level of AURKA in contrast to nocodazole (100 ng mL⁻¹). DLD-1 cells in different treatments were subjected to immunoblot analysis with different antibodies for detection of total AURKA, PCNA (loading control), histone H3 pSer10, and total histone H3. Representative immunoblot from at least two independent experiments is shown.