Fig. 4

Acetylation of CDK1 at K33 was regulated by HDAC3. a Heat map representing acetylation levels of proteins identified by acetyl-proteomics in PANC-1 cells after fisetin treatment. Red and blue indicated high and low acetylation levels, respectively. b KEGG pathway enrichment of proteins with differential expression acetylation after above treatment. c Heat maps of motif enrichment of amino acids upstream and downstream of all identified acetylated modification sites. Red and green represented that the amino acid was significantly enriched or reduced near the modification site, respectively. d, e Co-immunoprecipitation was used to detect the acetylation of CDK1 with fisetin treatment; Ac-Lys, Acetyl Lysine. f Immunoprecipitation was used to test the the deacetylation effect of HDAC3 to CDK1. g, h In situ proximity ligation assay (PLA) was used to determine the location and interaction between HDAC3 with CDK1-wild type and mutants. Top and side views of reconstructed 3D images of confocal micrographs were generated by Imaris software. Red spots represented positive interaction between HDAC3 and CDK1. Scale bars 10 μm. i Immunoprecipitation indicated that HDAC3 decreased the acetylation of CDK1 K33, and acetylation mimics of CDK1 K33 had highly binding capacity with HDAC3, while CDK1-K33R mutant impaired the interaction with HDAC3