flag-tagged hdac5 double mutant (hdac5 s259a/s498a)  (Addgene inc)

Journal: Science Advances

Article Title: Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis

doi: 10.1126/sciadv.ado7720

Figure Lengend Snippet: ( A ) Schematic workflow (left): HT29 cells were stably transfected with control (scrambled) or HDAC5 shRNA. Volcano plot of up-regulated acetylated peptides upon HDAC5 depletion (right). ( B ) HT29 cells were infected with indicated adenoviruses. Twenty-four hours after infection, immunoprecipitation (IP) followed by Western blotting was performed. HA, hemagglutinin. ( C ) Glutathione S -transferase (GST) pull-down assay was performed. Western blotting was then performed for the indicated proteins. ( D ) HT29 cells were treated with 5-fluorouracil (5FU; 10 μM) for indicated time points. Western blotting was then performed from nuclear and cytoplasmic extracts. ( E ) HT29 cells were treated with 5FU (10 μM) for indicated time points, and the nuclear extracts were subjected to immunoprecipitation followed by immunoblotting. ( F ) HT29 cells were treated with 5FU (10 μM) for indicated time points, and the nuclear extracts were subjected to immunoprecipitation. Western blotting was then performed. ( G and H ) HT29 control [Control; scrambled HDAC5 short hairpin RNA (shRNA)], HDAC5 knockdown (HDAC5kd; HDAC5 shRNA), and HDAC5 knockdown cells expressing HDAC5 mutant (HDAC5kd/HDAC5 S259/498A ) were subjected to proximity ligation assay (PLA) [(G), left]. Representative images are shown [(G), right]. Scale bars, 10 μm. The number of PLA foci per cell was quantified and plotted (H). Statistical analyses were done using two-way analysis of variance (ANOVA) (Bonferroni’s post hoc test). The data are representative of three independent experiments. Error bars are means ± SD. *** P < 0.001. ( I ) HT29 cells were transfected with an empty vector (EV) or the indicated FLAG-tagged PARP1 constructs. Twenty-four hours after transfection, nuclear extracts were subjected to immunoprecipitation. Western blotting was then performed (left). Schematic representation of the PARP1 full-length and deletion mutants (right). ( J ) HT29 cells were transfected as indicated. Twenty-four hours after transfection, nuclear extracts were subjected to immunoprecipitation. Western blotting was then performed (left). Schematic representation of the HDAC5 full-length and deletion mutants (right). (A), (G), (I), and (J) created with BioRender.com .

Article Snippet: The Flag-tagged HDAC5 double mutant (HDAC5 S259A/S498A ) was a gift from R, Shaw (Addgene, plasmid #32218).

Techniques: Stable Transfection, Transfection, Control, shRNA, Infection, Immunoprecipitation, Western Blot, Pull Down Assay, Knockdown, Expressing, Mutagenesis, Proximity Ligation Assay, Plasmid Preparation, Construct

Journal: Science Advances

Article Title: Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis

doi: 10.1126/sciadv.ado7720

Figure Lengend Snippet: ( A ) HT29 control (Control; scrambled HDAC5 shRNA) and HDAC5 knockdown (HDAC5kd; HDAC5 shRNA) cells were treated with 5FU (10 μM) for indicated time points. Western blotting was then performed for the indicated proteins from nuclear extracts. ( B ) HT29 cells were stably transfected (pooled zeomycin-resistant population) with control (Control; Scrambled p300 shRNA) or p300 knockdown (p300kd; p300 shRNA). These cells were treated with 5FU (10 μM) for indicated time points. Western blotting was then performed for the indicated proteins from nuclear extracts. ( C ) HT29 PARP1 and HDAC5 double knockdown (HDAC5kd/PARP1kd; HDAC5 and PARP1 shRNA) cells were transfected with FLAG-tagged wild-type PARP1, PARP1 K498R , PARP1 K505R , PARP1 K508R , PARP1 K521R , or PARP1 K532R constructs as indicated. Twenty-four hours after transfections nuclear extracts were prepared and subjected to immunoprecipitation followed by Western blotting for the indicated proteins. ( D ) AcK498-PARP1 and AcK521-PARP1 peptides were incubated either alone (Control) or in the presence of recombinant HDAC5. Mass spectrometry was then performed. The relative positions of acetylated Lys 498 (1068.60 Da) and Lys 521 (1389.79 Da), while deacetylated Lys 498 (1026.60 Da) and Lys 521 (1347.79 Da) PARP1 peptides are indicated. m/z , mass/charge ratio. ( E ) Results of HDAC5 deacetylation reactions using acetylated PARP1 peptides. ( F ) Schematic representation of the PARP1 domains. The lysine residues in the automodification domain are sites of HDAC5-mediated deacetylation. Created with BioRender.com . ( G ) The amino acid sequence encompassing human PARP1 K498 (red) and K521 (blue) residue. ( H ) HT29 control (Control; scrambled HDAC5 shRNA) and HDAC5 knockdown (HDAC5kd; HDAC5 shRNA) cells were treated with 5FU (10 μM) for indicated time points. Western blotting was then performed for the indicated proteins. ( I ) HT29 HDAC5 knockdown (HDAC5kd; HDAC5 shRNA) cells were stably transfected with Flag-tagged HDAC5 mutant constructs. These cells were treated with 5FU (10 μM) for indicated time points. Western blotting was then performed for the indicated proteins.

Article Snippet: The Flag-tagged HDAC5 double mutant (HDAC5 S259A/S498A ) was a gift from R, Shaw (Addgene, plasmid #32218).

Techniques: Control, shRNA, Knockdown, Western Blot, Stable Transfection, Transfection, Construct, Immunoprecipitation, Incubation, Recombinant, Mass Spectrometry, Sequencing, Residue, Mutagenesis

Journal: Science Advances

Article Title: Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis

doi: 10.1126/sciadv.ado7720

Figure Lengend Snippet: ( A ) Schematic representation of workflow to identify mutant PARP1 binding proteins upon 5FU (10 μM) treatment for 12 hours in HT29 cells (left). Volcano plot of proteins interacting with PARP1 mutant (PARP1 K521R ) (right). Created with BioRender.com . ( B ) HT29 PARP1 knockdown (PARP1kd; PARP1 shRNA) cells were transfected with EV or FLAG-tagged wild-type or mutant PARP1 constructs as indicated. Twenty-four hours after transfection, the cells were treated with 5FU (10 μM) for 12 hours. Immunoprecipitation followed by Western blotting for the indicated proteins was performed. ( C and D ) HT29 control (Control; Luc shRNA), HDAC5 knockdown (HDAC5kd; HDAC5 shRNA), and PARP1 knockdown cells (PARP1kd; PARP1 shRNA) expressing PARP1 mutants and HDAC5 knockdown cells expressing HDAC5 mutants were treated with 5FU (10 μM) for indicated time points. PLA was performed (C). Scale bars, 10 μm. The number of PLA foci per cell was quantified and plotted (D). Statistical analyses were done using two-way ANOVA (Bonferroni’s post hoc test). Error bars are means ± SD. ** P < 0.01; *** P < 0.001. ( E ) HT29 control (Control) and HDAC5 knockdown (HDAC5kd) cells were treated with 5FU (10 μM) for indicated time points. Western blotting was then performed. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( F ) HT29 control (Control; Luc shRNA), PARP1 knockdown cells expressing PARP1 K521R (PARP1 K521R ), as well as PARP1 and activating transcription factor 4 (ATF4) double knockdown cells (PARP1 and ATF4 shRNA) expressing PARP1 K521R (PARP1 K521R /ATF4kd) were treated with 5FU and subjected to RNA sequencing analysis. Red and blue indicate up-regulation and down-regulation, respectively. ( G ) HT29 control (Control; Luc shRNA), HDAC5 knockdown (HDAC5kd), PARP1 knockdown cells expressing PARP1 mutants, and HDAC5 knockdown cells expressing HDAC5 mutants were treated with 5FU (10 μM) for indicated time points. A reverse transcription quantitative polymerase chain reaction was performed. Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). Error bars are means ± SD of three independent experiments with triplicate samples. *** P < 0.001.

Article Snippet: The Flag-tagged HDAC5 double mutant (HDAC5 S259A/S498A ) was a gift from R, Shaw (Addgene, plasmid #32218).

Techniques: Mutagenesis, Binding Assay, Knockdown, shRNA, Transfection, Construct, Immunoprecipitation, Western Blot, Control, Expressing, RNA Sequencing Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Science Advances

Article Title: Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis

doi: 10.1126/sciadv.ado7720

Figure Lengend Snippet: ( A ) HT29 control (Control; Luc shRNA), HDAC5 knockdown (HDAC5kd; HDAC5 shRNA), PARP1 knockdown (PARP1kd; PARP1 shRNA), PARP1 knockdown cells expressing PARP1 K521R (PARP1 K521R ), as well as PARP1 and ATF4 double knockdown cells expressing PARP1 K521R (PARP1 K521R /ATF4kd) were treated with 5FU (10 μM) for 12 hours. The metabolites were extracted from the harvested cells and analyzed by liquid chromatography–mass spectrometry (LC-MS). Relative levels of specific metabolites normalized to cell number are shown in the heatmap. The heatmap depicts relative changes in intracellular metabolites. Orange and cyan indicate up-regulation and down-regulation, respectively. ( B ) Metabolic pathway enrichment analysis of (A). Metabolite data are representative of three independent experiments. ( C to G ) HT29 control (Control), HDAC5 knockdown (HDAC5kd), PARP1 knockdown (PARP1kd), PARP1 knockdown cells expressing PARP1 K521Q (PARP1 K521Q ) or PARP1 K521R (PARP1 K521R ), PARP1 and ATF4 double knockdown cells expressing PARP1 K521R (PARP1 K521R /ATF4kd), as well as HDAC5 knockdown cells expressing HDAC5 S259/498A (HDAC5kd/HDAC5 S259/498A ) were treated with 5FU (10 μM) for indicated time points. The cells were harvested, and metabolites were extracted. The relative amounts of the indicated metabolites were quantified using respective kits. Statistical analyses were done using two-way ANOVA (Bonferroni’s post hoc test). The data are representative of three independent experiments. Error bars are means ± SD. *** P < 0.001. ( H ) HT29 control (Control), HDAC5 knockdown (HDAC5kd), PARP1 knockdown (PARP1kd), as well as PARP1 knockdown cells expressing PARP1 K521Q (PARP1kd/PARP1 K521Q ) or PARP1 K521R (PARP1kd/PARP1 K521R ), PARP1 and ATF4 double knockdown cells expressing PARP1 K521R (PARP1 K521R /ATF4kd), and HDAC5 knockdown cells expressing HDAC5 S259/498A (HDAC5kd/HDAC5 S259/498A ) were treated with 5FU (10 μM) for indicated time points. The cellular oxygen consumption rate (OCR) was then measured. The data are representative of three independent experiments.

Article Snippet: The Flag-tagged HDAC5 double mutant (HDAC5 S259A/S498A ) was a gift from R, Shaw (Addgene, plasmid #32218).

Techniques: Control, shRNA, Knockdown, Expressing, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Science Advances

Article Title: Temporal regulation of acetylation status determines PARP1 role in DNA damage response and metabolic homeostasis

doi: 10.1126/sciadv.ado7720

Figure Lengend Snippet: ( A ) HT29 Luc2 control (Control; Luc shRNA); HDAC5 knockdown (HDAC5kd; HDAC5 shRNA); PARP1 knockdown (PARP1kd; PARP1 shRNA); PARP1 knockdown cells (PARP1kd; PARP1 shRNA) expressing PARP1 K498R (PARP1kd/PARP1 K498R ), PARP1 K521R (PARP1kd/PARP1 K521R ), or PARP1 K498R,521R (PARP1kd/PARP1 K498R,521R ); and HDAC5 knockdown cells expressing HDAC5 S259/498A (HDAC5kd/HDAC5 S259/498A ) were orthotopically injected into the cecum wall of nude mice. After 1 week, mice were administered 5FU (10 mg/kg) every alternate day. Olaparib was also coadministered at a dose of 10 mg/kg daily as indicated. Bioluminescence imaging was performed. ( B ) Bioluminescence quantification [(A) above]. The data are representative of three independent experiments using five individual mice per group. Error bars are means ± SD from five individual mice ( n = 5 mice per group). Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). *** P < 0.001. ( C and D ) At the end of 5 weeks, the liver and lung from indicated mice [(A) above] were analyzed by ex vivo imaging (C) and hematoxylin and eosin staining (D). ( E ) At the end of 5 weeks, the blood from mice in (A) above was used to isolate genomic DNA for examining circulating tumor cells. The data are representative of three independent experiments using five individual mice per group. Error bars are means ± SD from five individual mice ( n = 5 mice per group) * P < 0.05; ** P < 0.01; *** P < 0.001. ( F ) At the end of 5 weeks, lysates of primary orthotopic tumors from (A) above were analyzed by immunoblotting for the indicated proteins. ( G and H ) Representative image of immunostaining of the indicated proteins in different grades of colon adenocarcinoma and matched normal adjacent tissue (NAT) (G). Quantitation of HDAC5, AcK498-PARP1, and AcK521-PARP1 levels (H). Statistical analyses were done using one-way ANOVA (Dunn’s multiple comparison test). Error bars are means ± SD. ** P < 0.01; *** P < 0.001.

Article Snippet: The Flag-tagged HDAC5 double mutant (HDAC5 S259A/S498A ) was a gift from R, Shaw (Addgene, plasmid #32218).

Techniques: Control, shRNA, Knockdown, Expressing, Injection, Imaging, Ex Vivo, Staining, Western Blot, Immunostaining, Quantitation Assay, Comparison

Journal: Nature communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: Fig. 5 FAK-mediated class IIa HDACs tyrosine phosphorylation is regulated by FFSS. a Immunoprecipitation by anti-phosphotyrosine (referred to as “p- Y-1000” throughout) antibody. FFSS was performed for 10 min with Ocy454 cells. Phosphotyrosine immunoprecipitation was performed on cell lysates treated as indicated, and protein expression was determined by western blotting. FFSS reduced HDAC4 and HDAC5 with p-Y-1000 immunoprecipitation. b FLAG immunoprecipitation was performed in HDAC5-deficient cells stably expressing FLAG-HDAC5, subjected to FFSS for times as indicated. phospho- HDAC5 was decreased in a time-dependent manner. c HEK293T cells were transfected with FLAG-HDAC4, then treated for one hour as indicated with PF562271 (10 µM) or vanadate (1 mM) followed by phosphotyrosine immunoprecipitation and then immunoblotting. Tyrosine-phosphorylated HDAC4 was increased by vanadate treatment and decreased by FAK inhibitor treatment. P-values adjusted for multiple comparisons vs control are shown. d FAK kinase activity was measured by ADP-Glo kinase assay. E4Y1 (poly(Glu)/poly(Tyr) ratio of 4:1) polypeptides were used as a control substrate of FAK tyrosine kinase. Increased FAK kinase activity was detected when recombinant human HDAC5 protein was used as a substrate. e Recombinant human HDAC5 and E4Y1 (a control substrate) were incubated with ɣ-32P-ATP and FAK tyrosine kinase for 1 h, then separate by SDS-PAGE followed by autoradiography. FAK treatment showed ɣ-32P-ATP-positive band at the expected HDAC5 recombinant protein size. f Kinase assay reactions as in e were separated by SDS- PAGE followed by immunoblotting as indicated. CBB indicates Coomassie Brilliant Blue stain. One-way ANOVA followed by Tukey–Kramer post hoc test was used (d). Data are expressed as mean ± SEM. Each experiment was repeated three times (a–c, e, f). Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Phospho-proteomics, Immunoprecipitation, Expressing, Western Blot, Stable Transfection, Transfection, Control, Activity Assay, Kinase Assay, Recombinant, Incubation, SDS Page, Autoradiography, Staining

Journal: Nature communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: Fig. 7 RGD peptide blocks FAK activity and reduce Sost in a HDAC4/5-dependent manner. a Ocy454 cells were treated with cilengitide (10 µM and 50 µM) or PF562271 (10 µM) for 4 h, followed by RT-qPCR for FFSS-regulated genes as indicated. Both cilengitide and PF562271 regulate expression of FFSS-responsive genes. (n = 4 biologic replicates for RNA) P-values adjusted for multiple comparisons vs controls are shown. b Cells were treated with cilengitide (50 µM) for the indicated times followed by immunoblotting. c Cells were treated with the indicated doses of cilengitide (1 h) followed by immunoblotting. pHDAC4/5 immunoblotting was performed using an antibody that recognizes HDAC4 pS246 and HDAC5 pS259. d Saos2 cells were treated with cilengitide (100 µM) for 60 min followed by immunoblotting. e WT and HDAC4/5 double-knockout (H4H5-DKO) cells were treated with vehicle or cilengitide (50 µM) for 4 h followed by Sost RT-qPCR. Cilengitide treatment decreased Sost expression in control cells, but not in H4H5-DKO cells. (n = 4 biologic replicates for RNA) P-values vs control in the left and right panels. Two-sided unpaired t test e and one-way ANOVA followed by Tukey–Kramer post hoc test (a) were used. Data are expressed as mean ± SEM. Each experiment was repeated three times (b–d). Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Double Knockout, Control

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Journal: The Journal of Biological Chemistry

Article Title: Protein kinase D1 (PKD1) phosphorylation on Ser 203 by type I p21-activated kinase (PAK) regulates PKD1 localization

doi: 10.1074/jbc.M116.771394

Figure Lengend Snippet: Identification of a novel PAK/PKD/HDAC-signaling pathway. A, IEC-18 cells were transiently transfected with constructs encoding GFP-PKD1 or GFP-PKD1S203A. After 48 h the cultures were incubated in the absence (−) or presence of 10 nm ANG II for various times, as indicated. B, analysis of 70 cells at each time point. Results are expressed as the % of cells with GFP-PKD1 localized on the cell membrane. C, IEC-18 cells were transiently transfected with a plasmid encoding FLAG-tagged HDAC5. The cultures were incubated in the absence (−) or presence of 3.5 μm CRT0066101, 3.5 μm PF-3758309, 3.5 μm FRAX597, or 30 μm IPA-3 for 1 h before stimulation with 10 nm ANG II for 1 h. The cultures were then washed and fixed with 4% paraformaldehyde and stained with an antibody that detects the FLAG tag and Hoechst 33342 stain to visualize the nuclei. D, analysis of 100 cells for each treatment. Results are expressed as the % of cells with FLAG-HDAC5 localized in the nucleus. In each case the closed bars represent cultures stimulated with 10 nm ANG II. E, confluent cultures of IEC-18 cells were incubated in the absence (0) or in the presence of 3.5 μm CRT0066101 (CRT), 3.5 μm PF-3758309 (PF), 3.5 μm FRAX597 (FRAX), or 30 μm IPA-3 (IPA) for 1 h and then stimulated without (−) or with 10 nm ANG II for 15 min. F and G, IEC-18 cells were incubated in the absence (0) or in the presence of increasing concentrations of PF-3758309 (F) or FRAX597 (G) for 1 h and then stimulated without (−) or with 10 nm ANG II for 15 min. H, cultures of IEC-18 cells were transfected with non-targeting siRNA (Non. Targ) or with a mixture of siRNAs targeting PAK1 and PAK2 (siPAK1+2) for 5 days. Then the cultures were stimulated with 10 nm ANG II for 10 min. In E, F, G, and H, all incubations were terminated by the addition of 2× SDS-PAGE sample buffer, and cell lysates were resolved by SDS-PAGE. HDAC5 phosphorylation was determined by Western blot analysis using the antibody that detects its phosphorylated state on Ser498 and GAPDH as a loading control. J, GPCR signaling induces DAG accumulation at the plasma membrane, which mediates the translocation of inactive PKD1 from the cytosol to that cellular compartment. DAG also recruits and activates novel PKCs, which mediate the transphosphorylation of PKD1 on Ser744 (in mouse PKD1). DAG and PKC-mediated transphosphorylation of PKD1 act synergistically to promote PKD1 catalytic activation and autophosphorylation on Ser748. PAK1 and PAK2 also activated in response to GPCR signaling phosphorylate PKD1 at Ser203, thereby facilitating its dissociation from the membrane to the cytosol (PKDcyt) and to the nucleus (PKDnucleus), where PKD1 phosphorylates class IIa HDAC, including HDAC5 and HDAC7. In this manner the PAKs regulate class IIa HDAC phosphorylation and localization through phosphorylation of PKD1 on Ser203. Thus, our results identify a novel PAK/PKD/HDAC5 pathway in signal transduction. For more details, see “Discussion.”

Article Snippet: Cultures of IEC-18 cells were transfected with the plasmids containing a cDNA encoding either a GFP PKD WT or a GFP PKD S203A or an epitope (FLAG)-tagged-HDAC5 wild type from Addgene (catalog #32216) by using Lipofectamine 2000 (Invitrogen) as suggested by the manufacturer.

Techniques: Transfection, Construct, Incubation, Plasmid Preparation, Staining, FLAG-tag, SDS Page, Western Blot, Translocation Assay, Activation Assay, Transduction