12 research outputs found

    The Effects of Pharmacological Inhibition of Histone Deacetylase 3 (HDAC3) in Huntington’s Disease Mice

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    <div><p>An important epigenetic modification in Huntington’s disease (HD) research is histone acetylation, which is regulated by histone acetyltransferase and histone deacetylase (HDAC) enzymes. HDAC inhibitors have proven effective in HD model systems, and recent work is now focused on functional dissection of the individual HDAC enzymes in these effects. Histone deacetylase 3 (HDAC3), a member of the class I subfamily of HDACs, has previously been implicated in neuronal toxicity and huntingtin-induced cell death. Hence, we tested the effects of RGFP966 ((<i>E</i>)-N-(2-amino-4-fluorophenyl)-3-(1-cinnamyl-1<i>H</i>-pyrazol-4-yl)acrylamide), a benzamide-type HDAC inhibitor that selectively targets HDAC3, in the N171-82Q transgenic mouse model of HD. We found that RGFP966 at doses of 10 and 25 mg/kg improves motor deficits on rotarod and in open field exploration, accompanied by neuroprotective effects on striatal volume. In light of previous studies implicating HDAC3 in immune function, we measured gene expression changes for 84 immune-related genes elicited by RGFP966 using quantitative PCR arrays. RGFP966 treatment did not cause widespread changes in cytokine/chemokine gene expression patterns, but did significantly alter the striatal expression of macrophage migration inhibitory factor (<i>Mif)</i>, a hormone immune modulator associated with glial cell activation, in N171-82Q transgenic mice, but not WT mice. Accordingly, RGFP966-treated mice showed decreased glial fibrillary acidic protein (GFAP) immunoreactivity, a marker of astrocyte activation, in the striatum of N171-82Q transgenic mice compared to vehicle-treated mice. These findings suggest that the beneficial actions of HDAC3 inhibition could be related, in part, with lowered <i>Mif</i> levels and its associated downstream effects.</p></div

    Onset of Multiferroicity in Prototypical Single-Spin Cycloid BiFeO<sub>3</sub> Thin Films

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    In the room-temperature magnetoelectric multiferroic BiFeO3, the noncollinear antiferromagnetic state is coupled to the ferroelectric order, opening applications for low-power electric-field-controlled magnetic devices. While several strategies have been explored to simplify the ferroelectric landscape, here we directly stabilize a single-domain ferroelectric and spin cycloid state in epitaxial BiFeO3 (111) thin films grown on orthorhombic DyScO3 (011). Comparing them with films grown on SrTiO3 (111), we identify anisotropic in-plane strain as a powerful handle for tailoring the single antiferromagnetic state. In this single-domain multiferroic state, we establish the thickness limit of the coexisting electric and magnetic orders and directly visualize the suppression of the spin cycloid induced by the magnetoelectric interaction below the ultrathin limit of 1.4 nm. This as-grown single-domain multiferroic configuration in BiFeO3 thin films opens an avenue both for fundamental investigations and for electrically controlled noncollinear antiferromagnetic spintronics

    iPOND analysis reveals HDAC3 association with replication forks in Hut78 CTCL cells.

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    <p>Hut78 cells were pulsed for 15 minutes with EdU followed by either no thymidine chase or a 60 minute thymidine chase. The protein-DNA complexes were then cross-linked, nascent DNA was conjugated to biotin using click chemistry, and then protein-DNA complexes were purified using Streptavidin beads. The eluted proteins were then analyzed using western blot analysis. A no click reaction sample (No Clk) that did not include biotin azide was used as a negative control. 0.1% input samples were included for positive controls of each protein analyzed. PCNA served as a positive control for a replication fork bound protein and H2B served as a loading control and positive control for a chromatin bound protein.</p

    Dual treatment with RGFP966 and CTCL drugs has an additive effect on cell growth.

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    <p>Growth curves of dual treatment on HH cells or Hut78 cells. Cells were treated once at hour 0 with DMSO, 10 nM Depsipeptide (Depsi), 2 µM 966, or a combination of 2 µM 966 and either Bexarotene, Methotrexate, or ATRA. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (A) HH cells (left) or Hut78 cells (right) were treated with 20 µM or 75 µM Bexarotene alone or in combination with 966. (B) Cells were treated with 0.1 µM Methotrexate alone or in combination with 966. DMSO and 1 M Na<sub>2</sub>CO<sub>3</sub> served as vehicle controls. (C) Cells were treated with 2 µM ATRA alone or in combination with 966. ATRA was administered at hour 0 and re-dosed at 48 hours after treatment. For (A–C), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI, CTCL drug, or dual treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, Bexarotene: p = 0.003, and 966 plus Bexarotene: p = 0.002. For the Hut78 cells (right), Depsi: p = 0.001, 966: p = 0.08, Bexarotene: p = 0.01, and 966 plus Bexarotene: p = 0.009. (B) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, Methotrexate: p = 0.003, and 966 plus Methotrexate: p = 0.003. For the Hut78 cells (right) Depsi: p = 0.001, 966: p = 0.01, Methotrexate: p = 0.01, and 966 plus Methotrexate: p = 0.004. (C) HH cells (left), Depsi: p = 0.0008, 966: p = 0.003, ATRA: p = 0.002, and 966 plus ATRA: p = 0.0007. For the Hut78 cells (right) Depsi: p = 0.001, 966: p = 0.01, ATRA: p = 0.02, and 966 plus ATRA: p = 0.004.</p

    HDIs show selective inhibition of HDACs in CTCL cell lines.

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    <p>(A)Western blot analysis of whole cell lysates from Wild-type (WT) and <i>Hdac3</i>-null livers. Histones H3 and H4 served as loading controls. (B) Upper Panel: Western blot analysis of NIH 3T3 cells following treatment with various HDIs (indicated above each lane). Anti-histone H3 was used as a loading control. Lower panel: Western blot analysis of NIH 3T3 cells treated with either Trichostatin A (TSA) (1 µM), sodium butyrate (NaB) (5 mM), or increasing concentrations of nicotinamide (mM). (C) Western blot analysis of whole cell lysates prepared from cells that were transfected with either non-targeting siRNAs (NT) or siRNAs directed to the indicated Hdacs. (D) Western blot analysis of H3K56ac using whole cell lysates prepared from cells treated with the indicated amounts of RGFP966 for 24 hr. (E & F) Western blot analysis of (E) HH or (F) Hut78 cell lines treated with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, 10 µM 136, or 10 µM 966. Cells were treated for 24 hr and then harvested for protein isolation. Samples were run on the same gel and probed on the same membrane. Intervening lanes (represented by a black bar) were removed for side-by-side comparison of DMSO and Depsipeptide. Histones H3 and H4 were used as loading controls.</p

    CTCL cell lines are sensitive to pan and selective HDIs.

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    <p>(A) Growth curves of HDI treated HH cells (left) or Hut78 cells (right). Cells were treated once with DMSO, 10 nM Depsipeptide (Depsi), 10 µM 233, or 10 µM 966 at hour 0. Untreated cells and DMSO treated cells were used as controls. Cell growth was assessed at 0, 24, 48, and 72 hours after treatment. (B) Dose curves of 966 treated HH cells (left) and Hut78 cells (right). The experiment was performed in the same manner as (A) except that the cells treated were treated once with 2 µM, 5 µM, or 10 µM of 966 at hour 0. For both (A) and (B), representative curves are shown from experiments performed in triplicate that are consistent with other biological replicates. Statistical analysis was performed using a two-tail paired T-test and comparing the HDI treated cells to DMSO treated cells resulting in the following p values: (A) HH cells (left), Depsi: p = 0.0008, 233: p = 0.004, and 966: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 233: p = 0.006, and 966: p = 0.006. (B) HH cells (left), Depsi: p = 0.0008, 966 2 µM: p = 0.02, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006. For the Hut78 cells (right), Depsi: p = 0.002, 966 2 µM: p = 0.03, 966 5 µM: p = 0.01, and 966 10 µM: p = 0.006.</p
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