Fig. 4: Acetylome analysis revealed different acetylation profiles between CM-444/CM-1758 and the reference HDACi.

A Cell differentiation assay measuring CD11b and Sytox Green by flow cytometry in an ML-2 cell line treated with 25% GI₅₀ of CM-444 (260 nM), CM-1758 (210 nM) and the commercial HDACi Panobinostat (12.9 nM), Vorinostat (1.1 µM), Entinostat (2.3 µM), Quisinostat (18 nM) and Tubastatin (2.5 µM) for 48 h. The data shown are the mean of three biologically independent experiments. B Proteome and acetylome experimental design: ML-2 cells were treated with 25% GI₅₀ of CM-444 (260 nM) or CM-1758 (210 nM) and with reference HDACi Panobinostat (12.9 nM) or Vorinostat (1.1 µM) for 12 h. Then, cells were lysed and digested and the subsequent peptides were labeled and fractionated. 5 % of the fractions were kept for proteome analysis by LC-MS/MS. For the complete acetylome study, 95% of the obtained fractions were combined into five pools, and three consecutive IPs were performed for each pool, and the resulting samples were subsequently analyzed by LC-MS/MS. Figure 4/panel B, created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. C The total number of Acetyl-K sites quantified and the fraction of Acetyl-K sites regulated by each HDACi are shown. The bar chart shows the percentage of upregulated sites (log2 FC > 1, p < 0.05, shown in red) and downregulated sites (log2 FC < 1, p > 0.05, shown in blue). D Distribution of acetylated sites in the acetylated proteins. E Venn diagram of the total number of regulated Acetyl-K sites in ML-2 cells after treatment with CM-444, CM-1758, Panobinostat or Vorinostat compared with untreated cells. F PCA of acetylome data from ML-2 cells after treatment with CM-444, CM-1758, Panobinostat or Vorinostat compared with untreated cells. G Representation of Acetyl-K sites regulated after HDACi in histone and non-histone proteins. The data are shown as percentages, and the number of Acetyl-K sites regulated by each HDACi is indicated in the corresponding bar. Source data are provided as a Source data file.