Fig 1: BG45 reduced HDAC1 and HDAC2 protein expression in hippocampal neurons from APP/PS1 mice (n = 5). (A) Immunoblot analysis of levels of the HDAC1 and HDAC2 proteins in the hippocampus of mice treated with vehicle or BG45. (B) Quantification of the HDAC1 levels. (C) Quantification of the HDAC2 levels. Significant differences were determined using one-way ANOVA. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01, n = 5 mice per group.
Fig 2: Effect of BG45 on the expression of HDAC1, HDAC2, synaptic-related proteins after the HF-488-Aβ1–40 treatment. (A) Representative bands of the expression of HDAC1 and HDAC2 from control mice, HF-488-Aβ group and HF-488-Aβ + BG45 group. (B,C) Quantification of HDAC1 and HDAC2 is normalized to β-actin and is shown as a % of control. The expression of HDAC1, 2 was increased in the HF-488-Aβ group. Compared with the HF-488-Aβ group, the expression was significantly decreased in the HF-488-Aβ + BG45 group. (D) Representative bands of the expression of synapse-related protein from the control group, HF-488-Aβ group, and HF-488-Aβ + BG45 group. (E–G) Quantification of spinophilin, PSD-95, and synaptophysin (SYP) is normalized to β-actin and is shown as a % of control. The expression of spinophilin, PSD-95, and SYP was significantly decreased in the HF-488-Aβ group compared with the control group, while was improved in the HF-488-Aβ + BG45 group. n = 5. *p < 0.05, **p < 0.01.
Fig 3: HDAC3 indirectly regulated the expression of TNFAIP3 by inducing TNF-α. (A) RAW246.7 cells transfected with scr or HDAC3 siRNAs were treated or not with 1 µg/mL LPS for 60 and 240 min. Secreted TNF-α was detected in cell-free supernatants using ELISA. The results depict the mean ± SEM (n = 3). * p < 0.05 versus respective (-) using an unpaired t-test. (B) RAW246.7 cells transfected with scr or HDAC3 siRNAs were treated or not with 1 µg/mL LPS for 60 and 240 min with or without an anti-TNF-α-blocking antibody or recombinant murine TNF-α prior to mRNA extraction. The expression of TNFAIP3 was investigated at the mRNA level using RT-PCR. Data were normalized to the housekeeping RNA (18S) and expressed as the mean ± SEM (n = 3) of the percentage of production in LPS-stimulated scr cells. * p < 0.05 versus untreated scr or # p < 0.05 versus untreated HDAC3 siRNA using an unpaired t-test. Dots represent results of individual experiments.
Fig 4: HDAC3 knockdown partially recapitulated the effects of TSA. (A) RAW246.7 cells were transfected with the indicated siRNAs: target gene expression was evaluated using qPCR (upper panels) and expression at the protein level using Western blot analysis (lower panels). The results depict the percentage of target gene expression (mean ± SEM, n = 3) (upper panels) and a representative Western blot experiment; scr: scramble. Dots represent results of individual experiments. (B) siRNA-transfected RAW246.7 cells were treated with 1 µg/mL LPS for 60 min prior to mRNA extraction. The expression of TNF-α, IL-6, TNFAIP3 and CCL5 was investigated at the mRNA level using RT-PCR. Data were normalized to the housekeeping RNA (18S) and expressed as the mean ± SEM (n = 3) of the percentage of production in scr cells stimulated with LPS alone. As a comparison, cells transfected with scramble siRNA were also pre-treated with 100 ng/mL TSA. * p < 0.05 versus scr using an unpaired t-test; # p < 0.05 versus scr TSA using one-way ANOVA with Dunnett’s post hoc test. Dots represent results of individual experiments. (C) RAW246.7 cells transfected with scr or HDAC3 siRNA were treated with 1 µg/mL LPS for 60, 120 and 240 min with or without a 100 ng/mL TSA pre-treatment prior to mRNA extraction. The expression of TNF-α and TNFAIP3 was investigated at the mRNA level using RT-PCR. Data are normalized to the housekeeping RNA (18S) and expressed as the mean ± SEM (n = 3) of 2−ΔΔCt relative to (-). * p < 0.05 versus respective scr or # p < 0.05 versus respective HDAC3 siRNA using an unpaired t-test.
Fig 5: The effects of BG45 on Class I HDACs (HDAC1, 2, and 3). (A) The effect of BG45 on GFP-positive and APPsw-positive cell viability. (B) Immunoblot analysis of HDAC1, 2, and 3 levels in GFP- and APPsw-positive cells treated with vehicle or BG45 (15 μM) for different periods. (C) Quantification of HDAC1 levels. (D) Quantification of HDAC2 levels. (E) Quantification of HDAC3 levels. Significant differences were determined using Student’s t-test. All values are presented as the means ± SD from three independent experiments. * p < 0.05, ** p < 0.01.
from Cell Signaling Technology for Class I HDAC Antibody Sampler Kit