Fig. 4: NAT10 is lactylated by ATAT1.
A The iSLK-KSHV cells transduced with lentiviral NAT10 (NAT10-Flag) or its control (pCDH) were infected by lentiviral ESCO1 (ESCO1-Myc), ESCO2 (ESCO2-Myc), MYST1 (MYST1-Myc), and ATAT1 (ATAT1-Myc) for 48 h, and then subjected to anti-Flag immunoprecipitation. The interaction between NAT10 and acyltransferases was examined by Western blot with anti-Myc antibody. B The iSLK-KSHV cells transduced with lentiviral NAT10 (NAT10-Myc) or its control (pCDH) were infected by lentiviral ATAT1 (ATAT1-HA) for 48 h, and then subjected to anti-Myc immunoprecipitation to examine the lactylation level of NAT10 with anti-Pan Kla antibody. C Immunoprecipitation assay was performed to examine the interaction between endogenous NAT10 and ATAT1. D The iSLK-KSHV cells with ATAT1 (ATAT1-HA) overexpression were infected with lentiviral NAT10 (NAT10-Flag) or its control (pCDH) for 48 h, and then were employed to examine the co-localization of NAT10 and ATAT1 by immunofluorescence staining. Scar bars, 40 μm. E ATAT1-catalyzed NAT10 lactylation was determined by mixing soluble ATAT1, NAT10, and lactyl CoA (20 μΜ) in the in vitro lactylation assay. Western blot analysis was performed with the indicated antibodies. F The total RNAs from cells treated as in (B) were subjected to dot blot assay with anti-ac4C antibody. Methylene blue staining was used as the internal control. G The heatmap for differentially expressed tRNAs in iSLK-KSHV cells transduced with ATAT1 (ATAT1) or its control (pCDH) for 48 h by tRNA acRIP-seq analysis. The pseudo-color represented as the fold enrichment (IP/Input) of pCDH and ATAT1 group, respectively. Any tRNAs not detected in tRNA acRIP-seq were plotted as white in the heatmap. H The total RNAs from cells shown as in (G) were subjected to Northern blot with probes to tRNASer-CGA-1-1 and U6 as the control. I The cells treated as in (B) were subjected to the anti-Flag immunoprecipitation and analyzed by Western blot using anti-THUMPD1 antibody.
