Fig. 3: Chemotherapy induces the binding of NAT10 with ACLY in the nucleus.

A Venn diagram showed the common proteins between the nuclear proteins responsible for the production of acetyl-CoA including ACLY, ACSS2, pyruvate dehydrogenase (E1), dihydrolipoamide transacetylase(E2), dihydrolipoamide dehydrogenase (E3), as well as the tethering protein, E3-binding protein (E3BP) and the Flag-NAT10-binding proteins in HeLa cells [28]. The blue area illustrates the number of Flag-NAT10-binding proteins, the yellow area illustrates the number of nuclear proteins responsible for the production of acetyl-CoA, and the gray area illustrates the number of common proteins between two datasets. B Immunofluorescent staining showed the colocalization of ACLY (green) and NAT10 (red) treated with doxorubicin. Scale bar, 25 μm. C Cells were treated with doxorubicin. The cytoplasmic lysate and nuclear extraction were fractioned, which were subjected to immunoprecipitation using anti-NAT10 antibody. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies. Topoisomerase I (TOP I) and α-tubulin were used as nuclear and cytoplasmic marker, respectively. D GST pull-down was performed with purified Flag-NAT10 and GST-ACLY proteins. ACLY bound Flag-NAT10 was evaluated by WB using anti-Flag antibody. The amounts of GST fusion proteins and purified Flag-NAT10 protein used in the GST pull-down were shown by Coomassie blue staining. E GST pull-down was performed with purified His-ACLY and GST-NAT10’s deletion mutants. The amounts of GST fusion proteins and purified Flag-NAT10 protein used in the GST pull-down experiments were shown by Coomassie blue staining. NAT10-bound His-ACLY was evaluated by WB using the anti-His antibody. The schematic diagram represents the GST-NAT10 deletion mutant constructs [28] (lower panel). F GST pull-down was performed with purified Flag-NAT10 and GST-ACLY’s deletion mutants. The amounts of GST fusion proteins used in the GST pull-down were shown by Coomassie blue staining. ACLY bound Flag-NAT10 was evaluated by WB using the anti-Flag antibody. The schematic diagram represents the GST-ACLY deletion mutant constructs [64] (lower panel).