Fig 1: Enhanced lactylation of histone deacetylases contributes to the excessive expression of myeloid determining genes following Ppp2cα deletion. (A, C) Lin- cells were transfected with the lentiviral constructs encoding HDAC1-K412-Flag (K412), HDAC1-R412-Flag (R412), HDAC2-K451-Flag (K451) or HDAC2-R451-Flag (R451). (A) Overexpressed histone deacetylases (HDAC) were immunoprecipitated (IP) with anti-Flag antibody. The deacetylase activity of HDAC1 and HDAC2 was detected. N=3. P values were determined by one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± standard error of the mean (SEM). NS: not significant. (B) Schematic diagram of the structural domains of HDAC1 and HDAC2. (C) The impact of site-specific HDAC lactylation on the assembly of SIN3A/HDAC complex. Overexpressed HDAC proteins were IP with anti-Flag antibody, and associated proteins were detected by immunoblotting. Data are representative of 3 independent experiments. (D) LSK cells isolated from the bone marrow (BM) of tamoxifen-pretreated Ppp2caf/f (wild-type [WT]) and Ert2crePpp2caf/f (knockout [KO]) mice were subjected to chromatin immunoprecipitation-quantitative polymerase chain reaction analysis. The relative enrichment of H3K27ac at the promoter of target genes is shown. N=6. P values were determined by a two-tailed unpaired t test. Data are presented as mean ± SEM. ***P<0.001; ****P<0.0001. (E) LSK cells were prepared from the BM of tamoxifen-treated Ppp2caf/f (Pf/f) and Ert2crePpp2caf/f (EcrePf/f) mice, and then incubated with oxamate (Oxa). The acetylation level of histone H3 at lysine 27 (H3K27ac) was assessed by immunoblotting. β-actin was used as the internal loading control. Data are representative of 3 independent experiments. P values were determined by one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SEM. *P<0.05; ***P<0.001. (F) Immunoblot analysis of the LDHA and H3K27ac levels in the BM LSK cells from Ppp2caf/f, Ert2crePpp2caf/f, Ppp2caf/fLdhaf/+ and Ert2creErt2crePpp2caf/fLdhaf/+ mice.
Fig 2: Increased lactylation of HDAC1 and HDAC2 upon deletion of Ppp2cα. (A) LSK cells were prepared from the bone marrow (BM) of tamoxifen-pretreated Ppp2caf/f (wild-type [WT]) and Ert2crePpp2caf/f (knockout [KO]) mice. Statistical analysis of lactylated sites and proteins. (B) Heatmap depicting the increased lactylation levels of proteins involved in hematopoiesis. N=3. (C) Mass spectrometry analysis of the lactylated histone deacetylases (HDAC) peptides (HDAC1K412la and HDAC2K451la). ‘b’ refers to the amino-terminal parts of the peptide and ‘y’ refers to the carboxy-terminal parts of the peptide. Data represent 3 independent experiments. (D) The conservation of lactylated sites in HDAC1 and HDAC2. (E) Lin- cells isolated from the BM of tamoxifen-treated Ppp2caf/f (Pf/f) and Ert2crePpp2caf/f (EcrePf/f) mice were incubated with or without oxamate (Oxa). Subsequently, intracellular HDAC1 and HDAC2 were respectively immunoprecipitated (IP) with anti-HDAC1 and anti-HDAC2 antibodies, followed by detection of lactylation using anti-lactyllysine antibody (anti-Kla). (F) Immunoblot analysis assessing the pan-lysine acetylation (anti-Kac) levels in LSK. Coomassie blue staining (CB) indicates equal loading of protein. (G) Analysis of the site-specific lactylation of HDAC1 and HDAC2. Cell lysates from Lin- cells with HDAC1-Flag or HDAC2-Flag overexpressing were subjected to IP using an anti-Flag antibody (anti-Flag), followed by detection of lactylation with an anti-lactyllysine antibody (anti-Kla). The relative ratios of lactyl-HDAC to total-HDAC and total-HDAC to β-actin are shown. The experiments were repeated 3 times. P values were determined by a two-tailed unpaired t test. Data are presented as mean ± standard error of the mean. *P<0.05; NS: not significant.
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