Fig 1: Oral administration of simvastatin significantly recovers the reduced acetylation of histones H3/H4 and altered level of BDNF in AS mice brain. (A) Adult wild type and AS mice were orally administered simvastatin (1 mg/kg body weight) for 60 days, cortices were dissected out, lysed and subjected to immunoblotting using antibodies against Ube3a, HDAC1/2, histones H3/H4 and their acetylated derivatives and BDNF. (B–D) Quantitation of band intensities of HDAC1/2 (B), acetylated histones H3/H4 (C), and matured BDNF (D) as described in Figure 1. Each lane represents sample from the different mouse. Values plotted are mean ± SD with four mice in each treatment group. Data were examined by one-way ANOVA followed by Holm–Sidak post hoc test. The ‘a’ represent P < 0.01 compared to vehicle or simvastatin treated wild type mice group while “b” denote P < 0.01 with respect to vehicle treated AS mice group.
Fig 2: ZCWPW1 preserves H3K9ac by antagonizing HDAC’s deacetylation activity. a Immunoblotting of HDAC1, HDAC2, HDAC6, H3K9ac, and ZCWPW1 in HeLa cells transfected with the empty pCAG vector or with ZCWPW1-EGFP-pCAG at the indicated time points. b Immunoblot analysis of H3K9ac levels after overexpression of HDAC6 alone or co-expression of HDAC6 and ZCWPW1 in HeLa cells for 48 h. c Quantification of HDAC1, HDAC2, HDAC6, H3K9ac, and ZCWPW1 levels in HeLa cells transfected with empty pCAG vector or with ZCWPW1-EGFP-pCAG for 48 h. **P < 0.01, ***P < 0.001 by Student’s t test. Data represent the mean ± SEM from three independent experiments. d Inhibition of HDAC activity by ZCWPW1. HeLa cells were transfected with pCAG or ZCWPW1-EGFP-pCAG for 36 h, and the nuclear extracts were assessed with histone deacetylase activity assay kits according to the manufacturer’s instructions. Data are representative of three independent experiments
Fig 3: AS mice exhibit increased levels of HDAC1 and HDAC2 along with hypo-acetylation of histones H3(K9) and H4(K12) from early developmental days. (A) Cortical brain regions obtained from both wild type and AS mice at their different developmental stages (E16, P5, and P60) were processed for immunoblot analysis using antibodies against HDAC1 and HDAC2, various histones and their acetylated derivatives. (B–F) Densitometric analysis of the band intensity using ImageJ software. Band intensities of Ube3a (B), HDAC1 (C), and HDAC2 (D) were measured, divided by the band intensity of respective β-actin to normalize and expressed as fold change, while acetylated histones H3(K9) and H4(K12) were normalized against respective total histone (acetylated/total) and expressed as fold change. Values are mean ± SD with four animals in each group. The “a” indicate p < 0.05 compared to respective wild type group at E16, P5 and P60. Values were analyzed by two-way ANOVA with Holm–Sidak post-hoc test.
Fig 4: 4HR administration and histone deacetylase activity in Saos-2 cells. The administration of 4HR decreased histone deacetylase activity and the expression level of HDAC1, 3, 4, and 5. (A) Histone deacetylase activity decreased upon 4HR administration. When compared to untreated control, 10 and 100 μM 4HR group showed significantly lower values at 8 and 24 h after administration (* p < 0.05). (B) The level of acetylated histone 3 (Ac-H3) increased by 4HR administration. When Ac-H3 to H3 ratio of untreated control was compared, 4HR administered groups showed significantly higher values (* p < 0.05). (C) Expression level of HDAC1, 3, 4, and 5 decreased by 4HR administration. When compared to untreated control, 10 and 100 μM 4HR group showed significantly lower values at 8 and 24 h after administration (* p < 0.05). (D) Protein acetylation level generally increased by 4HR administration, as shown in whole cellular lysates.
Fig 5: ZCWPW1 physically interacts with multiple HDAC proteins. a Schematic diagram illustrating the experimental design for the RIME analysis. b Venn diagram showing the overlap of proteins binding to ZCWPW1 among the three repeats (Rep1, Rep2, and Rep3) in the RIME analysis. c Example of putative ZCWPW1-interacting proteins. The proteins are associated with chromatin regulation and DNA repair. d Co-IP analysis of ZCWPW1 binding proteins from PD14–PD16 testes protein extracts in WT and Zcwpw1–/– mice. HDAC1, HDAC2, and HDAC6 were immunoprecipitated with ZCWPW1. Data are representative of three independent experiments. e Co-IP analysis of ZCWPW1 binding proteins from PD14–PD16 testes protein extracts in WT mice. SMCHD1 was immunoprecipitated with ZCWPW1. Data are representative of three independent experiments. f Co-IP analysis of the interaction between ZCWPW1 and HDAC1, HDAC2, and HDAC6 in HeLa cells overexpressing ZCWPW1 for 48 h. Data are representative of three independent experiments
Supplier Page from Abcam for Histone Deacetylase (HDAC) Activity Assay Kit (Fluorometric)