Journal: Hypertension research : official journal of the Japanese Society of Hypertension

Article Title: Coupling factor 6 attenuates CXCR4 expression through the HIF-1α and c-Src pathways and promotes endothelial apoptosis and inflammation.

doi: 10.1038/hr.2014.65

Figure Lengend Snippet: Figure 2 Mechanism of coupling factor 6 (CF6)-induced decrease in CXC chemokine receptor type 4 (CXCR4) in human umbilical vein endothelial cells (HUVECs). (a) Chromatin immunoprecipitation (ChIP) assay performed with primers encompassing the hypoxia response element (HRE) in the presence or absence of CF6 at 107 M for 7 h. An anti-hypoxia-inducible factor (HIF)-1a antibody (dilution of 1:40) was used to immunoprecipitate the associated chromatin. (b) Histone deacetylase 3 (HDAC3) in immunoprecipitates using an anti-phospho-c-Src (pcSrc) antibody after a 30-min exposure of HUVECs to CF6 at 107 M (n ¼ 4). PcSrc after a 30-min exposure to CF6 at 107 M (n ¼ 4). HDAC3 and PcSrc were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the sample before immunoprecipitation. Po0.05 vs. CF6 ( ). (c) ChIP assay performed with primers encompassing the HRE in the presence or absence of CF6 at 107 M for 7 h. An anti-HDAC3 antibody (dilution of 1:40) was used to immunoprecipitate the associated chromatin.

Article Snippet: Human histone deacetylase 3 (HDAC3) antibody was obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Chromatin Immunoprecipitation, Histone Deacetylase Assay, Immunoprecipitation

Journal: PLoS ONE

Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

doi: 10.1371/journal.pone.0111277

Figure Lengend Snippet: A–F: Indicated amounts of HDAC3 inhibitors T247, T326, and T130 were added to C3 or C4 HeLa stable cell lines. The cells were harvested after 48 h of incubation and the fraction soluble in 1% Triton X-100 was subjected to western blot analysis (A–C). The insoluble fraction was subjected to a filter trap assay (D–E). Signals were detected by anti-GFP antibodies and chemiluminescence. Signal intensities were normalized to no inhibitors (DMSO only) = 100. The band from an anti-actin blot is shown as a loading control. Panels A, D: T247, B, E: T326, C, F: T130. *P≤0.05, **P≤0.01, ***P≤0.001 vs. 0×IC50 by ANOVA with multiple comparisons. N = 3. G, H: HDAC3 inhibitors do not increase Htt-ex1 mRNA levels. Effect of HDAC3 inhibitors on Htt-ex1 expression levels were assayed by qPCR. G: internal control = GAPDH, H: internal control = ACTB. Expression level was normalized to no inhibitor = 1.0. There was no statistical significance by ANOVA with multiple comparisons. N = 3.

Article Snippet: Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies).

Techniques: Stable Transfection, Incubation, Western Blot, TRAP Assay, Control, Expressing

Journal: PLoS ONE

Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

doi: 10.1371/journal.pone.0111277

Figure Lengend Snippet: A: Aspartate at the 166 th and 168 th amino acid of HDAC3 is crucial for its activity. An empty plasmid (–), FLAG tagged wild-type (wt), or D166A + D168A mutant (DA) of HDAC3 were overexpressed in 293T cells. After immunoprecipitation using anti-FLAG antibodies, pan-histone deacetylase activity was measured by fluorometric analysis. *P≤0.05 vs. empty plasmid by ANOVA and multiple comparisons. N = 3. Anti-FLAG and anti-actin western blots from cell lysates are shown below. B–C: HDAC3 overexpression reduces nuclear Htt-ex1 aggregates. Empty vector (–), FLAG-tagged wild-type or DA mutant HDAC3 were transfected to E3 and N3 cells. Amount of aggregate measured by filter trap assay are shown in B and C. *significant against – and DA by ANOVA and multiple comparisons. N = 3. D–G : Empty vector (–), FLAG-tagged wild-type or DA mutant HDAC3 were transfected to E3 and N3 cells. Cells harboring inclusion bodies are counted and their fraction in total cells was plotted in 2D and E. Representative GFP images of low powered magnification fields are shown in 2F and 2G. *significant against – and DA by ANOVA and multiple comparisons. H: HDAC3 shRNA reduces HDAC3 amount by 70%. Molecular weight markers are shown at the left side. I–J: HDAC3 knockdown increases nuclear aggregates. HDAC3 shRNA was transfected into E3 or N3 cells and the 1% TritonX-100 insoluble fraction was subjected to filter trap assay. *P = 0.0003 by t -test. N = 3.

Article Snippet: Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies).

Techniques: Activity Assay, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Histone Deacetylase Assay, Western Blot, Over Expression, Transfection, TRAP Assay, shRNA, Molecular Weight, Knockdown

Journal: PLoS ONE

Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

doi: 10.1371/journal.pone.0111277

Figure Lengend Snippet: A: HDAC3 inhibitors increase aggregated nuclear Htt-ex1. For filter trap analysis, three independently made insoluble fractions were analyzed on one single membrane; thus, there are error bars shown for 0×IC50s. *P≤0.05, ***P≤0.001 vs. each 0×IC50 by ANOVA and multiple comparisons. N = 3. B: HDAC3 inhibitors reduce cytoplasmic soluble Htt-ex1s. The effect of various HDAC inhibitors on 1% TritonX-100 soluble cytoplasmic Htt-ex1s. Indicated amount of HDAC inhibitors were added to E3 (cytoplasmic) or N3 (nuclear) cells for 48 h. Quantitated band intensity was normalized to each band without HDAC inhibitors (0×IC50); thus, there are no error bars. **P≤0.01, ***P≤0.001 vs. each 0×IC50 by ANOVA with multiple comparisons. N = 3. Anti-actin blots are shown for loading control.

Article Snippet: Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies).

Techniques: Membrane, Control

Journal: PLoS ONE

Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

doi: 10.1371/journal.pone.0111277

Figure Lengend Snippet: A: GST-HDAC3 binds directly to either cytoplasmic or nuclear Htt-ex1. GST pull-down assay of E1, E2, E3 (cytoplasm), and N1, N2, N3 (nuclear) HeLa cell lysates is shown. Pulled-down fraction was analyzed by anti-GFP or GST antibodies. *Non-specific band. B: HDAC3 immunoprecipitates almost exclusively with nuclear Htt-ex1s. E1, E3 (cytoplasm), N1, and N3 (nuclear) HeLa cells were transfected with FLAG-tagged HDAC3 and those lysates were immunoprecipitated with anti-FLAG antibodies immobilized to protein G agarose beads. The pre-IP fraction and the IPed fraction were analyzed using anti-FLAG or anti-GFP antibodies. Molecular weight markers are shown on the left. C: HDAC3 associates exclusively with nuclear inclusion bodies. E3 or N3 cells were fixed and stained with anti-HDAC3 antibodies and visualized by Alexa 546 conjugated secondary antibodies. Arrowheads: inclusion bodies with no HDAC3 signals associated. Arrows: HDAC3 signal-associated inclusion bodies. Bar = 20 µm.

Article Snippet: Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies).

Techniques: Pull Down Assay, Transfection, Immunoprecipitation, Molecular Weight, Staining

Journal: PLoS ONE

Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

doi: 10.1371/journal.pone.0111277

Figure Lengend Snippet: A–C: HDAC3 inhibitors inhibit proteasome activity. Three different HDAC3 inhibitors were added to HeLa cell cultures at the indicated concentrations. After 48 h of incubation, the proteasome activity of 5 µg total protein in a PBS lysate was measured using a fluorometric assay. A: T247, B: T326, C: T130. *P≤0.05 vs. each 0×IC50 by ANOVA with multiple comparisons. N = 3. D, E: HDAC3 inhibitors show a differential effect on cytoplasmic and nuclear proteasome activity. After incubating with the indicated amount of HDAC3 inhibitors for 48 h, cells were fractionated and the proteasome activity of 5 µg total protein from each fraction was independently measured. *P<0.05, **P<0.001 0×IC50 vs. 5×IC50 by t -tests N = 3. F: HDAC inhibitors have little or no direct proteasome inhibitory effect. Total protein (5 µg) from a HeLa cell PBS extract was subjected to the proteasome activity assay. During the incubation period for activity measurement, the indicated amount of HDAC inhibitors, or lactacystin as positive control, were added. Relative activity was shown with DMSO = 100%. ***P≤0.001 vs. DMSO by ANOVA with multiple comparisons. N = 3.

Article Snippet: Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies).

Techniques: Activity Assay, Incubation, Positive Control

Journal: PLoS ONE

Article Title: Differential Effect of HDAC3 on Cytoplasmic and Nuclear Huntingtin Aggregates

doi: 10.1371/journal.pone.0111277

Figure Lengend Snippet: A: HDAC3 activity is suppressed upon either cytoplasmic or nuclear Htt-ex1 expression. After two days of transfection in 293T cells, the HDAC3 activity of cellular lysates was measured using a fluorescence-based assay. B: Htt-ex1 overexpression does not alter pan-HDAC activity. After two days of transfection in 293T cells, the pan-HDAC activity was measured using a fluorescence-based assay.

Article Snippet: Visualization of the primary anti-HDAC3 antibody (Imgenex, San Diego, CA, USA). was done with the Alexa 546 secondary antibodies (Life Technologies).

Techniques: Activity Assay, Expressing, Transfection, Fluorescence, Over Expression

Journal: Molecular and Cellular Biology

Article Title: The N-CoR/Histone Deacetylase 3 Complex Is Required for Repression by Thyroid Hormone Receptor

doi: 10.1128/mcb.23.15.5122-5131.2003

Figure Lengend Snippet: FIG. 1. Unliganded TR specifically recruits N-CoR, HDAC3, and TBL1 to a repressed, stably integrated target gene. (a) Transcription assay. A (Gal4 5)-TK-luciferase plasmid was stably transfected into 293T cells as described in Materials and Methods. Effects of transfected Gal4 DBD or DBD fusion to the TR LBD (in the presence or absence of T3) are shown. Results are plotted as repression levels. (b) ChIP analysis of the experiment shown in panel a. (c) ChIP analysis of cells with stably integrated reporters transfected with a Gal4 DBD alone or fused to HDAC1 or HDAC3. Control IgG, control immunoglobulin G.

Article Snippet: Blots were visualized with ECL reagent (Amersham) and the following antibodies and dilutions: anti-N-CoR antibody (21) at 1:4,000, anti-SMRT antibody (12) at 1:500, anti-HDAC1 antibody at 1:1,000, anti-HDAC2 antibody at 1:2,000, and anti-HDAC4 antibody at 1:400 (Santa Cruz), and anti-HDAC3 antibodies (Novus) at 1:1,000.

Techniques: Stable Transfection, Transcription Assay, Luciferase, Plasmid Preparation, Transfection, Control

Journal: Molecular and Cellular Biology

Article Title: The N-CoR/Histone Deacetylase 3 Complex Is Required for Repression by Thyroid Hormone Receptor

doi: 10.1128/mcb.23.15.5122-5131.2003

Figure Lengend Snippet: FIG. 2. RevErb specifically recruits N-CoR, HDAC3, and TBL1 to a repressed, stably integrated target gene. (a) Transcription assay. The stable 293T cell line containing the (Gal4 5)-TK-luciferase reporter was transfected with Gal4 DBDs either alone or fused to RevErb LBD. Results are plotted as repression levels. (b) ChIP analysis of the ex- periment shown in panel a.

Article Snippet: Blots were visualized with ECL reagent (Amersham) and the following antibodies and dilutions: anti-N-CoR antibody (21) at 1:4,000, anti-SMRT antibody (12) at 1:500, anti-HDAC1 antibody at 1:1,000, anti-HDAC2 antibody at 1:2,000, and anti-HDAC4 antibody at 1:400 (Santa Cruz), and anti-HDAC3 antibodies (Novus) at 1:1,000.

Techniques: Stable Transfection, Transcription Assay, Luciferase, Transfection

Journal: Molecular and Cellular Biology

Article Title: The N-CoR/Histone Deacetylase 3 Complex Is Required for Repression by Thyroid Hormone Receptor

doi: 10.1128/mcb.23.15.5122-5131.2003

Figure Lengend Snippet: FIG. 3. Unliganded TR specifically recruits N-CoR and HDAC3 to a repressed, transiently transfected reporter gene. (a) Transcription assay. The (Gal4 5)-TK-luciferase reporter was transiently transfected with pCMX or pCMX-Gal4-TR LBDs into 293T cells. (b) ChIP analysis of the experiment shown in panel a. IgG, immunoglobulin G. (c) T3 dose response.

Article Snippet: Blots were visualized with ECL reagent (Amersham) and the following antibodies and dilutions: anti-N-CoR antibody (21) at 1:4,000, anti-SMRT antibody (12) at 1:500, anti-HDAC1 antibody at 1:1,000, anti-HDAC2 antibody at 1:2,000, and anti-HDAC4 antibody at 1:400 (Santa Cruz), and anti-HDAC3 antibodies (Novus) at 1:1,000.

Techniques: Transfection, Transcription Assay, Luciferase

Journal: Molecular and Cellular Biology

Article Title: The N-CoR/Histone Deacetylase 3 Complex Is Required for Repression by Thyroid Hormone Receptor

doi: 10.1128/mcb.23.15.5122-5131.2003

Figure Lengend Snippet: FIG. 6. Knockdown of HDAC3 markedly reduces repression by TR and RevErb. (a) Knockdown using SiRNA for HDAC1, -2, and -3. Shown are immunoblots for HDAC1 to -3 and HDAC4 (Control) after transfecting 293T cells with SiRNA for HDAC1, -2, and -3. (b) Transcription assay. The (Gal4 5)-TK-luciferase reporter was transiently transfected along with Gal4 DBD, Gal4-TR, or Gal4-RevErb into 293T cells treated with SiRNA for HDAC1 to -3 as described for panel a.

Article Snippet: Blots were visualized with ECL reagent (Amersham) and the following antibodies and dilutions: anti-N-CoR antibody (21) at 1:4,000, anti-SMRT antibody (12) at 1:500, anti-HDAC1 antibody at 1:1,000, anti-HDAC2 antibody at 1:2,000, and anti-HDAC4 antibody at 1:400 (Santa Cruz), and anti-HDAC3 antibodies (Novus) at 1:1,000.

Techniques: Knockdown, Western Blot, Control, Transcription Assay, Luciferase, Transfection