Journal: Nature Communications

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

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a WT, HDAC4-cKO ( HDAC4 flfl ;DMP1-cre ), HDAC5-KO, and H4H5-DKO mice were subjected in vivo cantilever bending of the right tibia. Each mouse underwent a 3-week regimen (3 days/week, 100 cycles/day, 2500 µε peak normal strain), and dynamic histomorphometry was performed on the tibia mid-shaft. Calcein labeling was performed at 2 days and 11 days prior to sacrifice. Exogenous loading significantly increased p.BFR in WT, HDAC4-cKO, and HDAC5 KO compared with contralateral tibiae. No significant p.BFR elevation observed in H4H5-DKO mice compared with contralateral tibiae ( n = 5–9 mice per group). p.SL (periosteal single-labeling surface), p.DL (periosteal double-labeling surface), pMAR (periosteal mineral apposition rate), p.MS (periosteal mineralizing surface), p.BFR (periosteal bone-mineral formation rate), WT (wild-type mice), H5KO (HDAC5 −/− ), H4KO (HDAC4 flfl ;DMP1-cre), DKO (H4H5-DKO, HDAC5 −/− ; HDAC4 fl/fl ;DMP1-cre). b Sclerostin immunohistochemistry (IHC) was performed in WT and H4H5-DKO mice ( n = 3). High-magnification images show representative images of sclerostin-positive and -negative cells in cortical bones. c All transverse sections were counted by ImageJ. Sclerostin-positive cells numbers are normalized by entire osteocyte number. ( n = 3 mice per group) P -values vs control (contralateral tibia). d qRT-PCR analyses from bone marrow-flushed tibias of WT and H4H5-DKO mice. Exogenous loading significantly reduced Sost mRNA expression in WT, but not H4H5-DKO mice. ( n = 4) P -values vs contralateral tibia. e , f Non-phospho (active) β-catenin IHC in WT and H4H5-DKO mice. Exogenous loading increased active β-catenin staining in periosteal cells of WT, but not in H4H5-DKO mice. Each experiment was repeated three times. ( n = 3 mice per group) P -values vs control (contralateral tibia) are shown in the figure. Two-sided unpaired t test was used ( a, c, d, f ). Data are expressed as mean ± SEM. 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: In Vivo, Labeling, Immunohistochemistry, Control, Quantitative RT-PCR, Expressing, Staining

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: a WT, HDAC4 KO, HDAC5-KO, and HDAC4/HDAC5 (H4H5) DKO Ocy454 cells were treated plus/minus FFSS for 3 h, followed by RT-qPCR for Sost . FFSS for 3 h reduced Sost expression in WT cells to a greater degree than in H4H5-DKO cells. P -values vs STATIC condition. Absolute SOST expression data are shown in the left panel ( n = 4 cells per group for control and DKO, n = 3 cells per group for H4KO and H5KO). The right panel shows the ratio of SOST comparing FFSS versus static-treatment within each cell line. P -values adjusted for multiple comparisons (versus control) are shown in the right panel. b , c HDAC5-deficient Ocy454 cells infected with FLAG-tagged HDAC5 were subjected to FFSS (3 h) followed by immunocytochemistry staining for HDAC4/5 and then confocal microscopy. Pictures show a representative image of each condition. HDAC5 (green, localization determined by anti-FLAG immunostaining), endogenous HDAC4 (red), and DAPI (blue). The nuclear densitometric intensity of FLAG-HDAC5 and endogenous HDAC4 were measured by ImageJ. P -values vs STATIC condition ( n = 15 cells for STATIC, n = 21 cells for HDAC5 FFSS, n = 29 cells for HDAC4 FFSS). Each experiment was repeated three times. d Ocy454 cells were subjected to FFSS for the indicated times and subjected to subcellular fractionation followed by immunoblotting. The nuclear fraction of endogenous HDAC5 and HDAC4 was quantified by densitometry over time. ( n = 3 biologic cell replicates were performed) P -values for each time point versus 0 min are shown in the right panel. e Volcano plot showing up- and downregulated genes by RNA-seq by FFSS treatment for 3 h in Ocy454 cells. Blue dots represent significantly regulated genes (log2 fold change (FFSS/STATIC) < −1 or >1, FDR < 0.05), black dots represent genes whose expression is not significantly regulated by FFSS. Sost gene was confirmed as a downregulated gene. f Control and HDAC4/5-deficient Ocy454 cells were treated plus/minus FFSS for 3 h followed by RNA-seq. Volcano plots as in e are shown. The majority of FFSS-induced DEGs are not regulated in cells lacking HDAC4/5. g Gene ontology analysis of FFSS-regulated genes showed enrichment of pathways linked to integrin/FAK signaling (matrix cellular components). In this graph, the x axis corresponds to the gene ontology enrichment score (adjusted P -value) for genes differentially expressed in response to FFSS. h Upstream analysis suggests that FAK and PYK2 are potential upstream candidate regulators of the coordinated gene expression changes seen by RNA-seq in response to FFSS. In this graph, the x axis corresponds to the upstream analysis score (adjusted P -value) for genes that were differentially expressed in response to FFSS. Listed in the figure are the FFSS-induced DEGs found in the upstream FAK-dependent “signature”. One-sided a, d and two-sided c unpaired t test, and one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc test ( a in the right panel) were used. Data are expressed as mean ± SEM. 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: Quantitative RT-PCR, Expressing, Control, Infection, Immunocytochemistry, Staining, Confocal Microscopy, Immunostaining, Fractionation, Western Blot, RNA Sequencing, Gene Expression

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: 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: 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: a , b Recombinant HDAC5 was phosphorylated in vitro by FAK followed by phospho-modification analysis by mass spectrometry. Tyrosine 642 on HDAC5 was phosphorylated in a FAK-dependent manner. Top panel: the total base peak chromatogram of the chymotrypsin digested HDAC5 in the absence of FAK treatment ( a ) or presence of FAK treatment ( b ). Bottom panel: extracted ion chromatogram for the m/z value (788.75) of the phosphorylated peptide KKLFSDAQPLQPLQVY#QAPL (# symbol represents phosphorylation) demonstrating the ability to detect the phosphorylated peptide upon FAK treatment ( b ). c 293T cells were transfected with FLAG-tagged WT and Y642F HDAC5 cDNAs followed by anti-FLAG immunoprecipitation and then elution with FLAG peptide. Eluted protein was then used as a substrate for in vitro kinase assays plus/minus recombinant FAK followed by immunoblotting as indicated. HDAC5 WT, but not Y642F mutant, is recognized by HDAC5 pY642 antibody. Each experiment was repeated three times. d Control and single-cell FAK-knockout cells were treated plus/minus FFSS (10 min) followed by immunoblotting as indicated. HDAC5 Y642 phosphorylation is reduced by FFSS, and dramatically reduced in FAK-mutant cells, as quantified in the bottom panel. ( n = 3 biologic replicates for protein were performed) P -values adjusted for multiple comparisons vs control in STATIC conditions are shown. e HDAC5-deficient Ocy454 cells were reconstituted with FLAG-tagged lentiviral constructs followed by immunoblotting as indicated. f Lentiviral reconstituted HDAC5-deficient cells were subjected to subcellular fractionation followed by immunoblotting as indicated. The percentage of the total FLAG-HDAC5 (WT or Y642F) in the nuclear fraction was measured by densitometry. ( n = 3 biologic replicates for protein were performed) P -values vs HDAC5 WT. g Cells as in f were subjected to anti-FLAG immunocytochemistry. Nuclear FLAG intensity was quantified ( n = 20 cells were analyzed). HDAC5 Y642F shows increased nuclear localization compared to HDAC5 WT grown under identical conditions. h Cells as in f were grown at 37 °C for 14 days. RNA was isolated for RT-qPCR. HDAC5-deficient cells show increased SOST expression which is reduced to a greater extent by HDAC5 Y642F than HDAC5 WT reconstitution. ( n = 4 biologic replicates for RNA for WT + eGFP and H5KO + eGFP) P -values (red) vs WT + eGFP. ( n = 5 biologic replicates for RNA for H5KO + H5WT and H5KO + H5Y642F) P -values (blue) vs H5KO + H5WT. Two-sided unpaired t test ( f , h ) and one-way ANOVA followed by Tukey–Kramer post hoc test ( d ) were used. Data are expressed as mean ± SEM. 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: Recombinant, In Vitro, Modification, Mass Spectrometry, Phospho-proteomics, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Control, Knock-Out, Construct, Fractionation, Immunocytochemistry, Isolation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

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

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: 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: Quantitative RT-PCR, Expressing, Western Blot, Double Knockout, Control

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 1. HDAC5 improved pathological symptoms in DHEA-induced polycystic ovary syndrome (PCOS) in mice. A. Brief flow chart of animal experiment. In young female mice, PCOS was induced by dehydroepiandrosterone (DHEA) administration (PCOS group). Some of the PCOS mice received injection of empty adenovirus (PCOS + Ad-vector group), whereas other PCOS mice received adenovirus expressing HDAC5 (PCOS + Ad-HDAC5 group). B. Ovarian morphology in each studied mouse group was determined by H&E staining at the magnification of 40×. Abbreviations: CL — corpus luteum; CF — cystic follicle. C–E. Serum levels of progesterone (C), testosterone (D) and estradiol (E) were detected by ELISA. F–H. mRNA and protein level of HDAC5 in ovary tissue of mice was measured by real-time PCR and Western blotting. Data was expressed as means ± SD (n = 6 in each group).

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Injection, Plasmid Preparation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 2. HDAC5 inhibited angiogenesis in ovaries of DHEA-induced PCOS mice. A. Relative mRNA levels of VEGFA in ova- ries from each group of mice. B–F. Contents of VEGFA (B), PDGFB (C), PDGFD (D), ANGPT1 (E) and ANGPT2 (F) in ovaries from each group of mice were determined by ELISA. G. The positive staining of CD31 was determined by immunohistochemistry. The allotypic serum was used as negative control to determine the specificity of CD31 staining. Magnification: 400×. Data was expressed as means ± SD (n = 6 in each group). Description of experimental groups as in the legend to Fig. 1. #, ## P < 0.05 and P < 0.01, respectively vs. control group; *, ** P < 0.05 and P < 0.01, respectively, vs. PCOS + Ad-vector group.

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Negative Control, Control, Plasmid Preparation

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 3. HDAC5 suppressed oxidative stress in ovaries of DHEA-induced PCOS mice. A. ROS level in each group was mea- sured by flow cytometric analysis using DCFH-DA probe. B–D. The malondialdehyde (MDA) levels (B), catalase (CAT) activity (C) and superoxide dismutase (SOD) activity (D) were measured as described in Methods. Data was expressed as means ± SD (n = 6 in each group). #, ## P < 0.05 and P < 0.01, respectively vs. control group; *, ** P < 0.05 and P < 0.01, respectively, vs. PCOS + Ad-vector group.

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Activity Assay, Control, Plasmid Preparation

Journal: Folia Histochemica et Cytobiologica

Article Title: HDAC5 inhibits ovarian angiogenesis in dehydroepiandrosterone-induced mouse model of polycystic ovary syndrome

doi: 10.5603/fhc.a2022.0024

Figure Lengend Snippet: Figure 4. HDAC5 inhibited HIF-1α/VEGF/VEGFR2 signaling in ovaries of DHEA-induced PCOS mice. A–B. Western-blot analysis of the protein levels of HIF-1α, VEGFA, p-VEGFR2 (Tyr 1175) and VEGFR2. C. Real-time PCR analysis of the mRNA levels of HIF-1α and VEGFR2. D. Co-immunoprecipitation was performed to determine the interaction between HDAC5 and VEGFR2 in ovaries of PCOS mice with anti-HDAC5 antibody. E. Acetylation level of VEGFR2 in ovaries from each group of mice was deter- mined by immunoprecipitation using anti-VEGFR2 antibody. F. Brief schematic presentation of the proposed effect of HDAC5 on angiogenesis. Data was expressed as means ± SD (n = 6 in each group). #, ## P < 0.05 and P < 0.01, respectively vs. control group;

Article Snippet: The supernatant was assigned to immunoblotting using Acetyl-Lysine antibody (1:500, A2391, ABclonal, Wuhan, China), VEGFR2 (1:500, sc-393163, Santa Cruz Biotech) and HDAC5 antibody (1:1000, 16166-1-AP, Proteintech Group).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Control

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 1. b1-AR/PKA activation reduces HDAC5 phosphorylation. A, Time course of HDAC5 dephos- phorylation by 10 nmol/L isoproterenol (ISO). Representative Western blots and grouped data from 4 independent experiments. Data obtained for each phosphorylation site were analyzed by 1-way ANOVA followed by Dunnett’s multiple comparisons tests. *P<0.05 vs 0 minutes. B, ARVM were treated with 10 nmol/L ISO or vehicle control (CON) for 10 minutes in the presence of 100 nmol/L CGP-20712A (CGP; a b1-AR-selective antagonist), 100 nmol/L ICI 118,551 (ICI; a b2-AR-selective antagonist), or vehicle (VEH). Representative Western blots and grouped data from 6 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons tests for each phosphorylation site. *P<0.05 vs VEH. C, ARVM were treated with 500 lmol/L N6-benzoyl cAMP (BNZ) to selectively activate PKA or vehicle control (CON) for 30 minutes. Representative Western blots and grouped data from 5 independent experiments (n=4 for S498 site). Unpaired t test for each phosphorylation site. *P<0.05 vs CON. b1-AR indicates b-adrenergic receptor; ARVM, adult rat ventricular myocytes; BNZ, N6-benzoyl cAMP; cTnI, cardiac troponin I; HDAC5, histone deacetylase 5; PKA, protein kinase A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 2. b-adrenergic stimulation induces nuclear accumulation of GFP-HDAC5. A, 3-Dimensional projections of an ARVM expressing GFP-HDAC5 at baseline and 45 minutes after the addition of 10 nmol/L isoproterenol (ISO). Z-stacks (25 z-sections at 1.5-lm intervals) were acquired as described in the Methods section. The fluorescent signals from the Cell Tracker Orange and DRAQ5 dyes were used to define the cytoplasmic (cyto) and nuclear (nuc) volumes for objective quantification of the average fluorescence intensity (F) arising from the GFP-HDAC5 signal within those compartments. B and C, Quantification of Fnuc and Fcyto following treatment with 10 nmol/L ISO or vehicle control (CON) for 15 to 45 minutes. All measurements obtained at the 15- and 45-minute time points were normalized to the baseline value for each cell (denoted by the dotted line). Two-way repeated-measures ANOVA followed by Sidak’s multiple comparisons tests. *P<0.05 vs CON at the same time point. †P<0.05 vs the same treatment group at the 15-minute time point. n=17 to 22 cells from 3 hearts per group. D, ARVM were fractionated into Triton X- 100-soluble (SOL) and -insoluble (INSOL) fractions via centrifugation following treatment with 10 nmol/L ISO or CON for 45 minutes. Representative Western blots show the distribution of GFP-HDAC5, GAPDH (cytosolic protein), and histone 2B (nuclear protein). Quantitative data show GFP-HDAC5 abundance in soluble and insoluble fractions from 5 independent experiments. In each experiment, the GFP-HDAC5 signal in CON and ISO fractions was normalized to the respective input. Unpaired t tests. *P<0.05 vs CON. ARVM indicates adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Expressing, Control, Centrifugation, Western Blot, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 3. ISO-induced dephosphorylation and nuclear accumulation of HDAC5 is PKA-dependent. A and B, Quantification of the nuclear/ cytoplasmic fluorescence ratio (Fnuc/Fcyto) in ARVM treated with 10 nmol/L ISO or CON in the presence of 10 lmol/L H89 (PKA inhibitor) or vehicle (VEH). Fnuc/Fcyto ratios were normalized to the baseline value obtained for each cell to account for differences in the basal distribution of GFP-HDAC5 between cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05 vs CON within the same pretreatment group (VEH/H89). †P<0.05 vs VEH within the same treatment group (CON/ISO). n=17 to 30 cells from 3 hearts per group. C, Quantification of Fcell at the 45-minute time point, normalized to baseline. No significant differences were detected by 2-way ANOVA. D, ARVM were treated with 10 nmol/L ISO or CON in the presence of 10 lmol/L H89 or vehicle (VEH). Representative Western blots and grouped data from 5 independent experiments. Unpaired t test. *P<0.05 vs VEH. ARVM indicates adult rat ventricular myocytes; CON, control; cTnI, cardiac troponin I; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; ISO, isoproterenol; PKA, protein kinase A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: De-Phosphorylation Assay, Western Blot, Control, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 4. The phosphorylation status of S259/S498, and not S279, is the key determinant of GFP-HDAC5 localization in ARVM. A and B, Fnuc/Fcyto and Fcell for ARVM expressing wildtype (WT) or nonphosphorylatable variants (S259/498A, S279A) of GFP-HDAC5. All individual data points, as well as the median and interquartile range, are shown. Kruskal-Wallis 1-way analysis of ranks followed by Dunn’s multiple comparisons test. *P<0.05, n=69 to 98 cells from 3 to 6 hearts per group. C, Western blot showing comparable expression of GFP-HDAC5 variants in ARVM. D, ARVM expressing GFP-HDAC5 variants or GFP alone were cotransduced with adenoviruses encoding a MEF2-luciferase reporter. ARVM were lysed and the luminescence readings from 25 lg protein were normalized to an internal control (ARVM expressing the luciferase reporter but not GFP/GFP-HDAC5). One-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05 vs GFP, †P<0.05 vs WT. n=6 to 7 independently transduced samples from 2 hearts per group. E and F, ARVM expressing WT or nonphosphorylatable GFP-HDAC5 variants were treated with 10 nmol/L isoproterenol (ISO) or vehicle control (CON) for 45 minutes. Fnuc/Fcyto ratios and Fcell values were normalized to the baseline value obtained for each cell to account for differences in the basal distribution of GFP-HDAC5 between cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05 vs CON for the same GFP-HDAC5 variant. †P<0.05 vs WT ISO, ‡P<0.05 vs S259/498A ISO. n=17 to 40 cells from 3 hearts per group. ARVM indicates adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; MEF, myocyte enhancing factor.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Phospho-proteomics, Expressing, Western Blot, Luciferase, Control, Variant Assay, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 5. PP2A dephosphorylates HDAC5 downstream of b-AR activation. ARVM were treated with 10 nmol/L isoproterenol (ISO) or vehicle control for 10 minutes in the presence of vehicle (VEH), 30 or 100 nmol/L okadaic acid (OKA). Representative Western blots and grouped data from 5 independent experiments. One-way ANOVA followed by Dunnett’s multiple comparisons tests for each phosphorylation site. *P<0.05 vs VEH. b-AR indicates b-adrenergic receptor; ARVM, adult rat ventricular myocytes; cTnI, cardiac troponin I; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; PLN, phospholamban; PP2A, protein phosphatase 2A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: Activation Assay, Control, Western Blot, Phospho-proteomics, Histone Deacetylase Assay

Journal: Journal of the American Heart Association

Article Title: β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

doi: 10.1161/jaha.116.004861

Figure Lengend Snippet: Figure 6. B55a targets PP2A to HDAC5 for dephosphorylation. A, ARVM expressing GFP or GFP-HDAC5 were treated with 10 nmol/L isoproterenol (ISO) or vehicle control (CON) for 10 minutes. GFP/GFP-HDAC5-containing complexes were immunoprecipitated from cell lysates, separated by SDS-PAGE and the resulting Western blots probed for B55a, PP2AC, and PP2AA. Representative Western blots and grouped data from 4 independent experiments. Unpaired t tests. *P<0.05 vs CON. B, NRVM expressing GFP-HDAC5 were transfected with scrambled (Scr) or PPP2R2A (B55a) siRNAs for 48 hours prior to treatment with 1 lmol/L ISO or CON for 60 minutes. Representative Western blots and grouped data from 4 independent experiments. Two-way ANOVA following by Sidak’s multiple comparisons tests. For B55a: *P<0.05 vs Scr within the same treatment group (CON/ISO). For S259: *P<0.05 vs CON within the same siRNA group (Scr/B55a). AdV indicates adenovirus; ARVM, adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; NRVM, neonatal rat ventricular myocytes; PP2A, protein phosphatase 2A.

Article Snippet: The beads were pelleted and the cleared supernatants incubated with HDAC5 antibody (Cell Signaling 20458; 1:70) or water (no antibody control) with gentle rocking at 4°C overnight.

Techniques: De-Phosphorylation Assay, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Transfection, Histone Deacetylase Assay

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 1. Expression and compartmental distribution of HDAC5 mu- tants in COS7 cells and 3T3-L1 adipocytes. A, schematic representation of HDAC5 wild type and FLAG-tagged truncated mutants used. B, 50 g of nuclear or cytosolic extracts from COS-7 cells transiently overexpressing empty vector (Mock) or truncated FLAG-tagged HDAC5 were analyzed by SDS-PAGE and immunoblotted (IB) with FLAG or HDAC5 antibodies. The arrow indicates HDAC5.

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Expressing, Plasmid Preparation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 2. Effect of truncation of the first 121 amino acids of HDAC5 (- 121) or truncation of the acetylase domain (-AD) on GLUT4 promoter activity in vitro. 3T3-L1 adipocytes 6 days after differentiation were tran- siently transfected with luciferase reporter plasmids 895-hG4-luc (firefly luciferase, for promoter activity) and pRLTK (Renilla luciferase, for transfec- tion efficiency), with and without plasmids encoding GEF, MEF2A, and wild type HDAC5 (wt HDAC5), -121-HDAC5, or -acetylase domain (AD) HDAC5. , inclusion in transfection. The means S.E. from three independent experiments are shown; the data were analyzed by a one-way analysis of co-variance, and statistically significant (p 0.05) changes are indicated.

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Activity Assay, In Vitro, Transfection, Luciferase

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 3. MEF and GEF interact with HDAC5 independently. A, 3T3-L1 adipocytes 6 days after differentiation were transiently transfected with luciferase reporter plasmids 895-hG4-luc (firefly luciferase, for promoter activity) and pRLTK (Renilla luciferase, for transfection efficiency), without or with plasmids encoding GEF, MEF2A, and wild type HDAC5 (wt HDAC5) or -MEF2 domain (-MEF) HDAC5. , inclusion in transfection. The data from three independent experiments are shown, and statistical significance was determined by a one-way analysis of co-variance. B, co-immunoprecipitation (IP) of HDAC5 with either MEF2A or GEF was performed in whole cell extracts from COS-7 cells transiently transfected with either MEF2A or GEF in the presence of -121- HDAC5 or -MEF2-HDAC5. The extracts were incubated with either preloaded normal mouse IgG-protein A/G beads or anti-FLAG beads overnight at 4 °C. The samples were then washed and analyzed by Western blot for the indicated protein. C, ratio of co-immunoprecipitated MEF2A or GEF to FLAG-tagged HDAC5 mutants. The means S.E. from four independent experiments are shown; statistical analysis was performed by a Student’s t test.

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Transfection, Luciferase, Activity Assay, Immunoprecipitation, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 4. Distribution of endogenous HDAC5 during differentiation of adipocytes. A, 3T3-L1 preadipocytes or adipocytes 6 days after differentia- tion were stained with an anti-HDAC5 antibody, then stained with an Alexa 568-conjugated secondary antibody (red), and fixed in the presence of DAPI staining (green) to mark the location of the nucleus. B, a ratio of Alexa 568 signal to DAPI signal was used to determine the relative amount of HDAC5 present in the nucleus. Data from at least eight cells were analyzed and ex- pressed as the means S.E. The data were analyzed by a Student’s t test.

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Staining

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 5. Phenylephrine treatment increases endogenous GLUT4 mRNA levels in preadipocytes with and without transactivating fac- tors. A, 50 g of COS7 whole cell extract transiently transfected with hu- man HDAC5 or 120 g of nuclear extracts of 3T3-L1 preadipocytes treated with or without phenylephrine for the indicated time periods and analyzed by SDS-PAGE and immunoblotting for HDAC5. The arrow indicates the ex- pected appearance of HDAC5. Con, control; IB, immunoblot. B, 3T3-L1 prea- dipocytes were transiently transfected with transactivating plasmids encod- ing GEF, MEF2A, and LXR. , inclusion in transfection. The cells were treated with 10 M phenylephrine for 24 h, and the RNA was isolated and analyzed by RT-PCR. The means S.E. from at least three independent ex- periments are shown; the data were analyzed by a Student’s t test.

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Transfection, SDS Page, Western Blot, Control, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 6. siRNA-mediated knockdown of class II HDACs increase endogenous GLUT4 mRNA in preadipocytes with and without transactivating factors. A, nuclear extracts of 3T3-L1 preadipocytes transiently transfected with scrambled siRNA or HDAC5 siRNA and analyzed by SDS-PAGE and immuno- blotting for endogenous HDAC5. The arrow indicates the HDAC5 band. IB, immunoblot. B, preadipocytes were transiently transfected with either scramble siRNA or the indicated specific HDAC siRNA in combination. RNA was isolated and subjected to quantitative PCR to determine the relative effectiveness of knocking down HDAC4, HDAC5, and HDAC9. C, preadipocytes were transiently transfected with either scrambled siRNA or HDAC-specific siRNA as indi- cated by . The cells were then incubated for 3 days and then transiently transfected again with either pcDNA3 (empty vector) or transactivator plasmids (MEF2A, GEF, and LXR). RNA was isolated and analyzed by RT-PCR for endogenous GLUT4 mRNA. The data are from at least three independent experi- ments. The data are expressed as the means S.E. and analyzed by one-way analysis of co-variance. The asterisk indicates a statistically significant differ- ence (p 0.05) without transactivating factors. The number sign indicates a statistically significant difference from knocking down all three class II HDACs and the addition of transactivating factors (p 0.05) over all other conditions. D, preadipocytes or adipocytes 6 days after differentiation were transiently transfected with either scrambled siRNA or HDAC-specific siRNA as indicated by . The cells were then incubated for 3 days and then transiently trans- fected again with either pcDNA3 (empty vector) or transactivator plasmids (MEF2A, GEF, and LXR). RNA was isolated and analyzed by RT-PCR for endoge- nous PPAR mRNA. The data are from at least three independent experiments. The data are expressed as the means S.E. and analyzed by one-way analy- sis of co-variance. The asterisk indicates a statistically significant increase in differentiated adipocyte PPAR compared with all other conditions (p 0.001).

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Knockdown, Transfection, SDS Page, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Incubation, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 7. The inhibition of HDAC5 is dependent on GEF and MEF2A binding to the GLUT4 promoter. 3T3-L1 preadipocytes were transiently trans- fected with either scrambled siRNA (control) or HDAC4/5/9-specific siRNA and incubated for 3 days and then transiently transfected again with luciferase reporter plasmids 895-hG4-luc or -hG4-luc (a loss of function mutation of Domain I (GEF binding site) and the MEF2-binding domain) (firefly luciferase, for promoter activity) and pRLTK (Renilla luciferase, for transfection efficiency), with and without plasmids encoding GEF, MEF2A, and wild type HDAC5 ( indicates inclusion in transfection). The means S.E. from three independent experiments are shown; the data were analyzed by a one-way analysis of co- variance. Statistically significant changes are indicated as follows: a, a statistical difference between scrambled and siRNA treated similar conditions; b, a statistical difference between 895-hG4-luc and the -hG4-luc cells treated with siRNA; and c, a statistical difference over all other conditions (p 0.005).

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Inhibition, Binding Assay, Control, Incubation, Transfection, Luciferase, Mutagenesis, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Class II Histone Deacetylases Limit GLUT4 Gene Expression during Adipocyte Differentiation

doi: 10.1074/jbc.m110.157107

Figure Lengend Snippet: FIGURE 8. Three class II HDACs are expressed in preadipocytes and can specifically regulate GLUT4 promoter activity in vitro. A, 120 g of nu- clear extracts of 3T3-L1 preadipocytes or adipocytes 6 days after differentia- tion were analyzed by SDS-PAGE and immunoblotted (IB) for endogenous HDAC4, HDAC5, or HDAC9 as indicated. Both the full-length protein and the lower splice variant were detected for all HDACs, but as the cells differenti- ated, the nuclear HDAC levels severely diminished. B, 3T3-L1 adipocytes 6 days after differentiation were transiently transfected with luciferase re- porter plasmids 895-hG4-luc (firefly luciferase, for promoter activity) and pRLTK (Renilla luciferase, for transfection efficiency), with and without plas- mids encoding GEF, MEF2A, wild type human HDAC4 (hHDAC4), wild type human HDAC5 (hHDAC5), or wild type human HDAC9 (hHDAC9). , inclu- sion in transfection. The means S.E. from three independent experiments are shown; the data were analyzed by a one-way analysis of co-variance, and statistically significant (p 0.005) changes are indicated.

Article Snippet: Total protein concentrations were determined with Coomassie Plus protein assay reagent (Thermo Scientific) according to the manufacturer’s protocol. siRNA Transfections—Experiments using siRNA were transfected in a similar manner as described above, with the following modifications: a final concentration of 50 M of either scrambled negative control siRNA (#5, Ambion, AM4638) or predesigned siRNA specifically for HDAC4, HDAC5, and HDAC9 purchased from Santa Cruz Biotechnology (sc-35541, sc-35543, and sc-35551).

Techniques: Activity Assay, In Vitro, SDS Page, Variant Assay, Transfection, Luciferase

Journal: The FEBS journal

Article Title: The myokine meteorin-like (metrnl) improves glucose tolerance in both skeletal muscle cells and mice by targeting AMPKα2.

doi: 10.1111/febs.15301

Figure Lengend Snippet: Fig. 5. Metrnl increased GLUT4 expression by stimulating HDAC5 phosphorylation. (A) Total mRNA from C2C12 myoblasts was prepared after metrnl (100 ngmL1) treatment for the indicated times, and real-time qRT-PCR was performed using GLUT4-specific primers, with b- actin mRNA as the positive control. (B) C2C12 myoblasts were treated with metrnl (100 ngmL1) for the indicated times. The cell lysates were analyzed by western blotting using anti-GLUT4 antibody, with b-actin as the control. (C) Time-dependent phosphorylation of HDAC5 after metrnl treatment. C2C12 myoblasts were incubated with metrnl (100 ngmL1) for the indicated times. Cell lysates were analyzed by western blotting using anti-phospho-HDAC5 (Thr498) antibody, with HDAC5 as the control. (D) C2C12 myoblasts were pre-treated with compound C (10 lM) and then treated with metrnl (100 ngmL1). Cell lysates were analyzed by western blotting using anti-phospho-HDAC5 (Thr498) antibody, with HDAC5 as the control. (E) C2C12 myoblasts were transiently transfected with AMPKa2 siRNA or non-target siRNA. Cell lysates were analyzed by western blotting using antibodies against phospho-HDAC5 (Thr498), AMPKa2, and HDAC5, with b-actin as the controls. (F) C2C12 myoblasts were treated with metrnl (100 ngmL1). Cytosolic and nuclear proteins were extracted from the cells. HDAC5 phosphorylation was evaluated by western blot analysis, with HDAC5 as the control. Western blotting was performed on nuclear and cytosolic fractions to detect nuclear (lamin B) and cytosolic (a-tubulin) marker proteins. (G) Representative images of phospho-HDAC5 treated with metrnl for 30 min. Scale bars, 10 lm (n = 5). (H) C2C12 myoblasts were immunoprecipitated with anti-14-3-3 antibody, followed by western blotting using anti-phospho-HDAC5, HDAC5, and 14-3-3 antibodies. (I) Representative images (phospho-HDAC5 and 14- 3-3 objective images) of cells treated with metrnl for 1 h. Scale bars, 10 lm (n = 5). (J) The relative occupancy of HDAC5 and AcH3 on the GLUT4 promoter was assessed using a ChIP analysis following 60 min of metrnl (100 ngmL1) treatment. The ChIP data represent the ratio of IP values for each region relative to the input. The results shown are from three independent experiments. Other results are displayed as the mean SEM of five experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: HDAC5 was immunoprecipitated using an HDAC5 antibody (Novus Biologicals, Littleton, CO, USA) or an 2099The FEBS Journal 287 (2020) 2087–2104 a 2020 The Authors.

Techniques: Expressing, Phospho-proteomics, Quantitative RT-PCR, Positive Control, Western Blot, Control, Incubation, Transfection, Marker, Immunoprecipitation