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hdac5 wild type wt  (Addgene inc)


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    Addgene inc hdac5 wild type wt
    Gene co-expression analysis of <t>HDAC5</t> and HDAC9 in the human substantia nigra. (A,B) Graphs showing the correlation between (A) HDAC5 and (B) HDAC9 and three markers of midbrain dopaminergic neurons ( TH, GIRK2/KCNJ6, ALDH1A1 ) in the human substantia nigra ( n = 101). The r and Bonferroni-corrected p -values shown on each graph. Raw data were derived from data set GSE60863 and analyzed using the R2 microarray platform. (C) Venn diagram showing the number of genes in the human substantia nigra displaying a multiple testing adjusted correlation of ≥0.6 with HDAC5 ( n = 1303) and HDAC9 ( n = 2361), and the overlap between them ( n = 651) in the SN. (D) Table showing a gene ontology (GO) enrichment analysis of these gene lists. The top category associated with each gene list is shown, along with the fold-enrichment and FDR adjusted p value.
    Hdac5 Wild Type Wt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hdac5 wild type wt/product/Addgene inc
    Average 91 stars, based on 5 article reviews
    hdac5 wild type wt - by Bioz Stars, 2026-05
    91/100 stars

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    1) Product Images from "Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling"

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2019.00191

    Gene co-expression analysis of HDAC5 and HDAC9 in the human substantia nigra. (A,B) Graphs showing the correlation between (A) HDAC5 and (B) HDAC9 and three markers of midbrain dopaminergic neurons ( TH, GIRK2/KCNJ6, ALDH1A1 ) in the human substantia nigra ( n = 101). The r and Bonferroni-corrected p -values shown on each graph. Raw data were derived from data set GSE60863 and analyzed using the R2 microarray platform. (C) Venn diagram showing the number of genes in the human substantia nigra displaying a multiple testing adjusted correlation of ≥0.6 with HDAC5 ( n = 1303) and HDAC9 ( n = 2361), and the overlap between them ( n = 651) in the SN. (D) Table showing a gene ontology (GO) enrichment analysis of these gene lists. The top category associated with each gene list is shown, along with the fold-enrichment and FDR adjusted p value.
    Figure Legend Snippet: Gene co-expression analysis of HDAC5 and HDAC9 in the human substantia nigra. (A,B) Graphs showing the correlation between (A) HDAC5 and (B) HDAC9 and three markers of midbrain dopaminergic neurons ( TH, GIRK2/KCNJ6, ALDH1A1 ) in the human substantia nigra ( n = 101). The r and Bonferroni-corrected p -values shown on each graph. Raw data were derived from data set GSE60863 and analyzed using the R2 microarray platform. (C) Venn diagram showing the number of genes in the human substantia nigra displaying a multiple testing adjusted correlation of ≥0.6 with HDAC5 ( n = 1303) and HDAC9 ( n = 2361), and the overlap between them ( n = 651) in the SN. (D) Table showing a gene ontology (GO) enrichment analysis of these gene lists. The top category associated with each gene list is shown, along with the fold-enrichment and FDR adjusted p value.

    Techniques Used: Expressing, Derivative Assay, Microarray

    HDAC5 and HDAC9 transcripts and protein are expressed in dopaminergic neurons in mouse substantia nigra. (A,B) RT-qPCR showing the expression of transcripts for (A) Hdac5 and (B) Hdac9 in mouse midbrain at postnatal day (P)5 and P90 relative to the levels of the geometric mean of three reference mRNAs, Gapdh , Sdha , and Hprt1 . Data are mean ± SEM from n = 3–6 mice at each time point. ∗∗ P < 0.01, Student’s t -test. (C) Quantification of TH-immunopositive neurons in the SN that express HDAC5 or HDAC9. (D,E) Immunohistochemistry showing (D) HDAC5 (red) and (E) HDAC9 (red) expression in TH-positive neurons (green; arrow), colabelled with DAPI (blue), in adult mouse substantia nigra. Scale bar = 50 μm. Data are mean ± SD from n = 3 mice.
    Figure Legend Snippet: HDAC5 and HDAC9 transcripts and protein are expressed in dopaminergic neurons in mouse substantia nigra. (A,B) RT-qPCR showing the expression of transcripts for (A) Hdac5 and (B) Hdac9 in mouse midbrain at postnatal day (P)5 and P90 relative to the levels of the geometric mean of three reference mRNAs, Gapdh , Sdha , and Hprt1 . Data are mean ± SEM from n = 3–6 mice at each time point. ∗∗ P < 0.01, Student’s t -test. (C) Quantification of TH-immunopositive neurons in the SN that express HDAC5 or HDAC9. (D,E) Immunohistochemistry showing (D) HDAC5 (red) and (E) HDAC9 (red) expression in TH-positive neurons (green; arrow), colabelled with DAPI (blue), in adult mouse substantia nigra. Scale bar = 50 μm. Data are mean ± SD from n = 3 mice.

    Techniques Used: Quantitative RT-PCR, Expressing, Immunohistochemistry

    siRNAs targeting HDAC5 or HDAC9 , but not other class-IIa HDAC family members HDAC4 or HDAC7 , promote neurite growth in SH-SY5Y cells. (A) Representative photomicrographs of SH-SY5Y cells immunocytochemically stained for HDAC5 (red) or HDAC9 (red) with DAPI (blue). Scale bar = 10 μm. (B) Representative photomicrographs of SH-SY5Y cells and (C) graph of neurite length at 24, 48, and 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9). Scale bar = 50 μm. Data are mean ± SEM as percentage of the siSCR control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. siSCR group; two-way ANOVA with post hoc Tukey’s test. (D) Immunocytochemistry showing nuclear localization of mutant HDAC5-S259A/S498A which is retained in the nucleus. Graphs of (E) AcH3-K9.K14 levels and (F,G) neurite length of SH-SH5Y cells at 72 h post-transfection with a control plasmid (GFP) or plasmids expressing wild-type (WT) HDAC5 or mutant HDAC5-S259A/S498A. Scale bar = 100 μm. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05 vs. Control; one-way ANOVA with post hoc Fishers LSD test.
    Figure Legend Snippet: siRNAs targeting HDAC5 or HDAC9 , but not other class-IIa HDAC family members HDAC4 or HDAC7 , promote neurite growth in SH-SY5Y cells. (A) Representative photomicrographs of SH-SY5Y cells immunocytochemically stained for HDAC5 (red) or HDAC9 (red) with DAPI (blue). Scale bar = 10 μm. (B) Representative photomicrographs of SH-SY5Y cells and (C) graph of neurite length at 24, 48, and 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9). Scale bar = 50 μm. Data are mean ± SEM as percentage of the siSCR control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. siSCR group; two-way ANOVA with post hoc Tukey’s test. (D) Immunocytochemistry showing nuclear localization of mutant HDAC5-S259A/S498A which is retained in the nucleus. Graphs of (E) AcH3-K9.K14 levels and (F,G) neurite length of SH-SH5Y cells at 72 h post-transfection with a control plasmid (GFP) or plasmids expressing wild-type (WT) HDAC5 or mutant HDAC5-S259A/S498A. Scale bar = 100 μm. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05 vs. Control; one-way ANOVA with post hoc Fishers LSD test.

    Techniques Used: Staining, Transfection, Control, Immunocytochemistry, Mutagenesis, Plasmid Preparation, Expressing

    Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in SH-SY5Y cells and dopamine neurons in E14 rat VM primary cultures. Graphs showing (A) the relative levels of acetylated histone 3 (Ach3), (B) neurite length and (C) lactate dehydrogenase (LDH) levels as a measure of cell viability in SH-SY5Y cells treated with 0.01 or 0.1 μM MC1568 for 72 h. (D,E) Graphs showing (D) the relative levels of acetylated histone 3 (Ach3) and (E,F) neurite length in TH-positive neurons in primary cultures of E14 rat VM treated with 0.01 μM MC1568 for 24 h. (G) Graph showing neurite length and (H) representative photomicrographs of primary cultures of E14 rat VM at 24 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) or HDAC9 (siHDAC9). Scale bar = 50 μm. Insert are tracing of individual neurons in the image. Data are mean ± SEM of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 vs. siSCR group; Student’s t -test or one-way ANOVA with post hoc Fishers LSD test.
    Figure Legend Snippet: Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in SH-SY5Y cells and dopamine neurons in E14 rat VM primary cultures. Graphs showing (A) the relative levels of acetylated histone 3 (Ach3), (B) neurite length and (C) lactate dehydrogenase (LDH) levels as a measure of cell viability in SH-SY5Y cells treated with 0.01 or 0.1 μM MC1568 for 72 h. (D,E) Graphs showing (D) the relative levels of acetylated histone 3 (Ach3) and (E,F) neurite length in TH-positive neurons in primary cultures of E14 rat VM treated with 0.01 μM MC1568 for 24 h. (G) Graph showing neurite length and (H) representative photomicrographs of primary cultures of E14 rat VM at 24 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) or HDAC9 (siHDAC9). Scale bar = 50 μm. Insert are tracing of individual neurons in the image. Data are mean ± SEM of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 vs. siSCR group; Student’s t -test or one-way ANOVA with post hoc Fishers LSD test.

    Techniques Used: Inhibition, Transfection

    Class-IIa HDAC inhibition increases BMP2 and SMAD1 expression in SH-SY5Y cells. (A) Schema of the BMP pathway. (B–G) RT-qPCR data showing (B) BMP2 , (C) BMPR2 , (D) ACVR2A , (E) BMPR1B , (F) SMAD1 and (G) SMAD5 mRNA levels relative to the levels of the geometric mean of three reference mRNAs, GAPDH , TBP and B2M , in SH-SY5Y cells treated for 12 h with 0.1 μM of the class-IIa HDAC inhibitor MC1568. Data are mean ± SEM from n = 4 independent experiments expressed as fold change relative to the control. (H) Graph and (I) representative photomicrographs showing the expression of GFP (as a readout of Smad-dependent transcription) in SH-SY5Y cells transfected with a Smad-GFP reporter construct and co-transfected with either a scrambled siRNA, or siRNAs against HDAC5 or HDAC9 or treated with MC1568 for 72 h. (J) Graph showing the relative levels of pSmad1/5/8 in TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. Scale bar = 50 μm. Data are mean ± SEM from n = 3 independent experiments expressed as fold change relative to the control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; Student’s t -test or one-way ANOVA with post hoc Tukey’s test as appropriate).
    Figure Legend Snippet: Class-IIa HDAC inhibition increases BMP2 and SMAD1 expression in SH-SY5Y cells. (A) Schema of the BMP pathway. (B–G) RT-qPCR data showing (B) BMP2 , (C) BMPR2 , (D) ACVR2A , (E) BMPR1B , (F) SMAD1 and (G) SMAD5 mRNA levels relative to the levels of the geometric mean of three reference mRNAs, GAPDH , TBP and B2M , in SH-SY5Y cells treated for 12 h with 0.1 μM of the class-IIa HDAC inhibitor MC1568. Data are mean ± SEM from n = 4 independent experiments expressed as fold change relative to the control. (H) Graph and (I) representative photomicrographs showing the expression of GFP (as a readout of Smad-dependent transcription) in SH-SY5Y cells transfected with a Smad-GFP reporter construct and co-transfected with either a scrambled siRNA, or siRNAs against HDAC5 or HDAC9 or treated with MC1568 for 72 h. (J) Graph showing the relative levels of pSmad1/5/8 in TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. Scale bar = 50 μm. Data are mean ± SEM from n = 3 independent experiments expressed as fold change relative to the control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; Student’s t -test or one-way ANOVA with post hoc Tukey’s test as appropriate).

    Techniques Used: Inhibition, Expressing, Quantitative RT-PCR, Control, Transfection, Construct

    The neurite growth-promoting effects of HDAC5 inhibition require BMP-Smad signaling. (A–E) In all experiments cells were transfected with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) and neurite length was analyzed at 24 h. (A) Quantification of neurite length per cell (B) representative photomicrographs when co-treated with 1 μg/ml of the BMPR1 inhibitor, dorsomorphin. Neurites are indicated by white arrows. Scale bar = 50 μm. (C,D) Quantification of neurite length per cell and (E) representative photomicrographs of cells co-transfected with plasmids overexpressing (C) the inhibitory I-Smad, Smad7 or (D,E) Smad4 dominant negative (Smad4dn). (F) Graph showing the neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. (G) A rescue experiment in which the cells were transfected with the nuclear-restricted HDAC5 mutant and cultured with or without 50 ng/ml BMP2. Data mean ± SEM as a percentage of the control of n = 3–4 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.01 vs. siSCR group alone; one-way ANOVA with post hoc Tukey’s test.
    Figure Legend Snippet: The neurite growth-promoting effects of HDAC5 inhibition require BMP-Smad signaling. (A–E) In all experiments cells were transfected with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) and neurite length was analyzed at 24 h. (A) Quantification of neurite length per cell (B) representative photomicrographs when co-treated with 1 μg/ml of the BMPR1 inhibitor, dorsomorphin. Neurites are indicated by white arrows. Scale bar = 50 μm. (C,D) Quantification of neurite length per cell and (E) representative photomicrographs of cells co-transfected with plasmids overexpressing (C) the inhibitory I-Smad, Smad7 or (D,E) Smad4 dominant negative (Smad4dn). (F) Graph showing the neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. (G) A rescue experiment in which the cells were transfected with the nuclear-restricted HDAC5 mutant and cultured with or without 50 ng/ml BMP2. Data mean ± SEM as a percentage of the control of n = 3–4 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.01 vs. siSCR group alone; one-way ANOVA with post hoc Tukey’s test.

    Techniques Used: Inhibition, Transfection, Dominant Negative Mutation, Mutagenesis, Cell Culture, Control

    Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in cells overexpressing wild-type or A53T α-synuclein, or treated with MPP + . (A,B) Representative photomicrographs of SH-SY5Y cells transfected with (A) GFP or (B) an expression plasmid expressing GFP-tagged wild-type α-synuclein (αSynWT-GFP) and immunocytochemically stained for α-synuclein (red). (C,D) Graphs of neurite length of SH-SY5Y cells at 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9) and co-transfected with a GFP control plasmid or a plasmid expressing (C) αSynWT-GFP or (D) αSynA53T-GFP. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; ### p < 0.001 vs. siSCR plus WT or A53T α-synuclein: One-way ANOVA with Tukey’s post hoc test. (E) Graphs of neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 1 mM MPP+ with or without 0.01 μM of MC1568. Data are presented as mean ± SEM as a percentage of the control of n = 3 independent experiments. ∗∗ p < 0.01, vs. Control; One-way ANOVA with Fishers LSD post hoc test.
    Figure Legend Snippet: Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in cells overexpressing wild-type or A53T α-synuclein, or treated with MPP + . (A,B) Representative photomicrographs of SH-SY5Y cells transfected with (A) GFP or (B) an expression plasmid expressing GFP-tagged wild-type α-synuclein (αSynWT-GFP) and immunocytochemically stained for α-synuclein (red). (C,D) Graphs of neurite length of SH-SY5Y cells at 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9) and co-transfected with a GFP control plasmid or a plasmid expressing (C) αSynWT-GFP or (D) αSynA53T-GFP. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; ### p < 0.001 vs. siSCR plus WT or A53T α-synuclein: One-way ANOVA with Tukey’s post hoc test. (E) Graphs of neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 1 mM MPP+ with or without 0.01 μM of MC1568. Data are presented as mean ± SEM as a percentage of the control of n = 3 independent experiments. ∗∗ p < 0.01, vs. Control; One-way ANOVA with Fishers LSD post hoc test.

    Techniques Used: Inhibition, Transfection, Expressing, Plasmid Preparation, Staining, Control



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    Image Search Results


    Gene co-expression analysis of HDAC5 and HDAC9 in the human substantia nigra. (A,B) Graphs showing the correlation between (A) HDAC5 and (B) HDAC9 and three markers of midbrain dopaminergic neurons ( TH, GIRK2/KCNJ6, ALDH1A1 ) in the human substantia nigra ( n = 101). The r and Bonferroni-corrected p -values shown on each graph. Raw data were derived from data set GSE60863 and analyzed using the R2 microarray platform. (C) Venn diagram showing the number of genes in the human substantia nigra displaying a multiple testing adjusted correlation of ≥0.6 with HDAC5 ( n = 1303) and HDAC9 ( n = 2361), and the overlap between them ( n = 651) in the SN. (D) Table showing a gene ontology (GO) enrichment analysis of these gene lists. The top category associated with each gene list is shown, along with the fold-enrichment and FDR adjusted p value.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: Gene co-expression analysis of HDAC5 and HDAC9 in the human substantia nigra. (A,B) Graphs showing the correlation between (A) HDAC5 and (B) HDAC9 and three markers of midbrain dopaminergic neurons ( TH, GIRK2/KCNJ6, ALDH1A1 ) in the human substantia nigra ( n = 101). The r and Bonferroni-corrected p -values shown on each graph. Raw data were derived from data set GSE60863 and analyzed using the R2 microarray platform. (C) Venn diagram showing the number of genes in the human substantia nigra displaying a multiple testing adjusted correlation of ≥0.6 with HDAC5 ( n = 1303) and HDAC9 ( n = 2361), and the overlap between them ( n = 651) in the SN. (D) Table showing a gene ontology (GO) enrichment analysis of these gene lists. The top category associated with each gene list is shown, along with the fold-enrichment and FDR adjusted p value.

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Expressing, Derivative Assay, Microarray

    HDAC5 and HDAC9 transcripts and protein are expressed in dopaminergic neurons in mouse substantia nigra. (A,B) RT-qPCR showing the expression of transcripts for (A) Hdac5 and (B) Hdac9 in mouse midbrain at postnatal day (P)5 and P90 relative to the levels of the geometric mean of three reference mRNAs, Gapdh , Sdha , and Hprt1 . Data are mean ± SEM from n = 3–6 mice at each time point. ∗∗ P < 0.01, Student’s t -test. (C) Quantification of TH-immunopositive neurons in the SN that express HDAC5 or HDAC9. (D,E) Immunohistochemistry showing (D) HDAC5 (red) and (E) HDAC9 (red) expression in TH-positive neurons (green; arrow), colabelled with DAPI (blue), in adult mouse substantia nigra. Scale bar = 50 μm. Data are mean ± SD from n = 3 mice.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: HDAC5 and HDAC9 transcripts and protein are expressed in dopaminergic neurons in mouse substantia nigra. (A,B) RT-qPCR showing the expression of transcripts for (A) Hdac5 and (B) Hdac9 in mouse midbrain at postnatal day (P)5 and P90 relative to the levels of the geometric mean of three reference mRNAs, Gapdh , Sdha , and Hprt1 . Data are mean ± SEM from n = 3–6 mice at each time point. ∗∗ P < 0.01, Student’s t -test. (C) Quantification of TH-immunopositive neurons in the SN that express HDAC5 or HDAC9. (D,E) Immunohistochemistry showing (D) HDAC5 (red) and (E) HDAC9 (red) expression in TH-positive neurons (green; arrow), colabelled with DAPI (blue), in adult mouse substantia nigra. Scale bar = 50 μm. Data are mean ± SD from n = 3 mice.

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry

    siRNAs targeting HDAC5 or HDAC9 , but not other class-IIa HDAC family members HDAC4 or HDAC7 , promote neurite growth in SH-SY5Y cells. (A) Representative photomicrographs of SH-SY5Y cells immunocytochemically stained for HDAC5 (red) or HDAC9 (red) with DAPI (blue). Scale bar = 10 μm. (B) Representative photomicrographs of SH-SY5Y cells and (C) graph of neurite length at 24, 48, and 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9). Scale bar = 50 μm. Data are mean ± SEM as percentage of the siSCR control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. siSCR group; two-way ANOVA with post hoc Tukey’s test. (D) Immunocytochemistry showing nuclear localization of mutant HDAC5-S259A/S498A which is retained in the nucleus. Graphs of (E) AcH3-K9.K14 levels and (F,G) neurite length of SH-SH5Y cells at 72 h post-transfection with a control plasmid (GFP) or plasmids expressing wild-type (WT) HDAC5 or mutant HDAC5-S259A/S498A. Scale bar = 100 μm. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05 vs. Control; one-way ANOVA with post hoc Fishers LSD test.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: siRNAs targeting HDAC5 or HDAC9 , but not other class-IIa HDAC family members HDAC4 or HDAC7 , promote neurite growth in SH-SY5Y cells. (A) Representative photomicrographs of SH-SY5Y cells immunocytochemically stained for HDAC5 (red) or HDAC9 (red) with DAPI (blue). Scale bar = 10 μm. (B) Representative photomicrographs of SH-SY5Y cells and (C) graph of neurite length at 24, 48, and 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9). Scale bar = 50 μm. Data are mean ± SEM as percentage of the siSCR control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. siSCR group; two-way ANOVA with post hoc Tukey’s test. (D) Immunocytochemistry showing nuclear localization of mutant HDAC5-S259A/S498A which is retained in the nucleus. Graphs of (E) AcH3-K9.K14 levels and (F,G) neurite length of SH-SH5Y cells at 72 h post-transfection with a control plasmid (GFP) or plasmids expressing wild-type (WT) HDAC5 or mutant HDAC5-S259A/S498A. Scale bar = 100 μm. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05 vs. Control; one-way ANOVA with post hoc Fishers LSD test.

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Staining, Transfection, Control, Immunocytochemistry, Mutagenesis, Plasmid Preparation, Expressing

    Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in SH-SY5Y cells and dopamine neurons in E14 rat VM primary cultures. Graphs showing (A) the relative levels of acetylated histone 3 (Ach3), (B) neurite length and (C) lactate dehydrogenase (LDH) levels as a measure of cell viability in SH-SY5Y cells treated with 0.01 or 0.1 μM MC1568 for 72 h. (D,E) Graphs showing (D) the relative levels of acetylated histone 3 (Ach3) and (E,F) neurite length in TH-positive neurons in primary cultures of E14 rat VM treated with 0.01 μM MC1568 for 24 h. (G) Graph showing neurite length and (H) representative photomicrographs of primary cultures of E14 rat VM at 24 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) or HDAC9 (siHDAC9). Scale bar = 50 μm. Insert are tracing of individual neurons in the image. Data are mean ± SEM of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 vs. siSCR group; Student’s t -test or one-way ANOVA with post hoc Fishers LSD test.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in SH-SY5Y cells and dopamine neurons in E14 rat VM primary cultures. Graphs showing (A) the relative levels of acetylated histone 3 (Ach3), (B) neurite length and (C) lactate dehydrogenase (LDH) levels as a measure of cell viability in SH-SY5Y cells treated with 0.01 or 0.1 μM MC1568 for 72 h. (D,E) Graphs showing (D) the relative levels of acetylated histone 3 (Ach3) and (E,F) neurite length in TH-positive neurons in primary cultures of E14 rat VM treated with 0.01 μM MC1568 for 24 h. (G) Graph showing neurite length and (H) representative photomicrographs of primary cultures of E14 rat VM at 24 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) or HDAC9 (siHDAC9). Scale bar = 50 μm. Insert are tracing of individual neurons in the image. Data are mean ± SEM of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01 vs. siSCR group; Student’s t -test or one-way ANOVA with post hoc Fishers LSD test.

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Inhibition, Transfection

    Class-IIa HDAC inhibition increases BMP2 and SMAD1 expression in SH-SY5Y cells. (A) Schema of the BMP pathway. (B–G) RT-qPCR data showing (B) BMP2 , (C) BMPR2 , (D) ACVR2A , (E) BMPR1B , (F) SMAD1 and (G) SMAD5 mRNA levels relative to the levels of the geometric mean of three reference mRNAs, GAPDH , TBP and B2M , in SH-SY5Y cells treated for 12 h with 0.1 μM of the class-IIa HDAC inhibitor MC1568. Data are mean ± SEM from n = 4 independent experiments expressed as fold change relative to the control. (H) Graph and (I) representative photomicrographs showing the expression of GFP (as a readout of Smad-dependent transcription) in SH-SY5Y cells transfected with a Smad-GFP reporter construct and co-transfected with either a scrambled siRNA, or siRNAs against HDAC5 or HDAC9 or treated with MC1568 for 72 h. (J) Graph showing the relative levels of pSmad1/5/8 in TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. Scale bar = 50 μm. Data are mean ± SEM from n = 3 independent experiments expressed as fold change relative to the control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; Student’s t -test or one-way ANOVA with post hoc Tukey’s test as appropriate).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: Class-IIa HDAC inhibition increases BMP2 and SMAD1 expression in SH-SY5Y cells. (A) Schema of the BMP pathway. (B–G) RT-qPCR data showing (B) BMP2 , (C) BMPR2 , (D) ACVR2A , (E) BMPR1B , (F) SMAD1 and (G) SMAD5 mRNA levels relative to the levels of the geometric mean of three reference mRNAs, GAPDH , TBP and B2M , in SH-SY5Y cells treated for 12 h with 0.1 μM of the class-IIa HDAC inhibitor MC1568. Data are mean ± SEM from n = 4 independent experiments expressed as fold change relative to the control. (H) Graph and (I) representative photomicrographs showing the expression of GFP (as a readout of Smad-dependent transcription) in SH-SY5Y cells transfected with a Smad-GFP reporter construct and co-transfected with either a scrambled siRNA, or siRNAs against HDAC5 or HDAC9 or treated with MC1568 for 72 h. (J) Graph showing the relative levels of pSmad1/5/8 in TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. Scale bar = 50 μm. Data are mean ± SEM from n = 3 independent experiments expressed as fold change relative to the control. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; Student’s t -test or one-way ANOVA with post hoc Tukey’s test as appropriate).

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Inhibition, Expressing, Quantitative RT-PCR, Control, Transfection, Construct

    The neurite growth-promoting effects of HDAC5 inhibition require BMP-Smad signaling. (A–E) In all experiments cells were transfected with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) and neurite length was analyzed at 24 h. (A) Quantification of neurite length per cell (B) representative photomicrographs when co-treated with 1 μg/ml of the BMPR1 inhibitor, dorsomorphin. Neurites are indicated by white arrows. Scale bar = 50 μm. (C,D) Quantification of neurite length per cell and (E) representative photomicrographs of cells co-transfected with plasmids overexpressing (C) the inhibitory I-Smad, Smad7 or (D,E) Smad4 dominant negative (Smad4dn). (F) Graph showing the neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. (G) A rescue experiment in which the cells were transfected with the nuclear-restricted HDAC5 mutant and cultured with or without 50 ng/ml BMP2. Data mean ± SEM as a percentage of the control of n = 3–4 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.01 vs. siSCR group alone; one-way ANOVA with post hoc Tukey’s test.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: The neurite growth-promoting effects of HDAC5 inhibition require BMP-Smad signaling. (A–E) In all experiments cells were transfected with 25 nM of a scrambled siRNA (siSCR) or siRNAs against HDAC5 (siHDAC5) and neurite length was analyzed at 24 h. (A) Quantification of neurite length per cell (B) representative photomicrographs when co-treated with 1 μg/ml of the BMPR1 inhibitor, dorsomorphin. Neurites are indicated by white arrows. Scale bar = 50 μm. (C,D) Quantification of neurite length per cell and (E) representative photomicrographs of cells co-transfected with plasmids overexpressing (C) the inhibitory I-Smad, Smad7 or (D,E) Smad4 dominant negative (Smad4dn). (F) Graph showing the neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 0.01 μM MC1568. (G) A rescue experiment in which the cells were transfected with the nuclear-restricted HDAC5 mutant and cultured with or without 50 ng/ml BMP2. Data mean ± SEM as a percentage of the control of n = 3–4 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.01 vs. siSCR group alone; one-way ANOVA with post hoc Tukey’s test.

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Inhibition, Transfection, Dominant Negative Mutation, Mutagenesis, Cell Culture, Control

    Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in cells overexpressing wild-type or A53T α-synuclein, or treated with MPP + . (A,B) Representative photomicrographs of SH-SY5Y cells transfected with (A) GFP or (B) an expression plasmid expressing GFP-tagged wild-type α-synuclein (αSynWT-GFP) and immunocytochemically stained for α-synuclein (red). (C,D) Graphs of neurite length of SH-SY5Y cells at 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9) and co-transfected with a GFP control plasmid or a plasmid expressing (C) αSynWT-GFP or (D) αSynA53T-GFP. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; ### p < 0.001 vs. siSCR plus WT or A53T α-synuclein: One-way ANOVA with Tukey’s post hoc test. (E) Graphs of neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 1 mM MPP+ with or without 0.01 μM of MC1568. Data are presented as mean ± SEM as a percentage of the control of n = 3 independent experiments. ∗∗ p < 0.01, vs. Control; One-way ANOVA with Fishers LSD post hoc test.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

    doi: 10.3389/fcell.2019.00191

    Figure Lengend Snippet: Beneficial effects of pharmacological or siRNA-mediated inhibition of HDAC5 or HDAC9 in cells overexpressing wild-type or A53T α-synuclein, or treated with MPP + . (A,B) Representative photomicrographs of SH-SY5Y cells transfected with (A) GFP or (B) an expression plasmid expressing GFP-tagged wild-type α-synuclein (αSynWT-GFP) and immunocytochemically stained for α-synuclein (red). (C,D) Graphs of neurite length of SH-SY5Y cells at 72 h post-transfection with 25 nM of a scrambled siRNA (siSCR) or siRNAs against the four class-IIa HDACs (siHDAC4, siHDAC5, siHDAC7, siHDAC9) and co-transfected with a GFP control plasmid or a plasmid expressing (C) αSynWT-GFP or (D) αSynA53T-GFP. Data are presented as the mean ± SEM as a percentage of the GFP control of n = 3 independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. Control; ### p < 0.001 vs. siSCR plus WT or A53T α-synuclein: One-way ANOVA with Tukey’s post hoc test. (E) Graphs of neurite length of TH-positive neurons in primary cultures of E14 rat VM at 24 h post-treatment with 1 mM MPP+ with or without 0.01 μM of MC1568. Data are presented as mean ± SEM as a percentage of the control of n = 3 independent experiments. ∗∗ p < 0.01, vs. Control; One-way ANOVA with Fishers LSD post hoc test.

    Article Snippet: 1.5 × 10 cells per well were transfected using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with various combinations of the following plasmids or siRNAs as indicated in the relevant figure legends: EGFP-alpha-synuclein-WT (Addgene plasmid # 40822 ; RRID:Addgene_40822 ) and EGFP-alphasynuclein-A53T (Addgene plasmid #40823 ; RRID:Addgene_40823 ) were a gift from David Rubinsztein ( ); pCMV5 DPC4 (1-514) (Smad4 dominant negative; Addgene plasmid #14040; RRID:Addgene_14040 ) was a gift from Joan Massague ( ). pcDNA3-Smad7 was a gift from Aristidis Moustakas (Addgene plasmid #80893 ; RRID:Addgene_80893); GFP Cignal reporter (Qiagen CCS-017G); HDAC5 wild-type (WT) (Addgene plasmid #32213 ; RRID:Addgene_32213 ) and HDAC5 S259A/S498A (Addgene plasmid #32216 ; RRID:Addgene_32216 ) were gifts from Reuben Shaw ( ).

    Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Staining, Control