Journal: Immunity

Article Title: A cis-regulatory element controls expression of histone deacetylase 9 to fine-tune inflammasome-dependent chronic inflammation in atherosclerosis.

doi: 10.1016/j.immuni.2025.01.003

Figure Lengend Snippet: Figure 1. Targeted deletion of the CRE at Hdac9 exacerbates atherosclerosis (A) Risk variants at HDAC9 for human atherosclerotic phenotypes are located in the cis-regulatory element (CRE), which is conserved across species with enrichment of active histone marks.23 Shown are all the common variants ±5 kb of the lead signal rs2107595 for coronary artery disease (CAD), large artery stroke (LAS), peripheral artery disease (PAD), and atherosclerotic aortic calcifications (AACs) from the respective GWASs. Hdac9DCRE mice lack 1.1 kb of the CRE. See also Figures S1A and S1B. (B–E) Hdac9DCREApoe/ and control Hdac9+/+Apoe/ mice received a chow diet for 28 weeks. (B) Representative oil red O-stained aortic root plaques (indicated by arrowheads). (C) Quantification of aortic root plaque sizes. n = 7 mice per genotype. Two-sided unpaired t test. (D) Representative Mac2 immunostaining. (E) Quantification of macrophage area. n = 7 mice per genotype. Two-sided unpaired t test. Scale bars, 200 mm in (B) and (D). See also Figures S1C–S1K. (F and G) Hdac9DCREApoe/ and control Hdac9+/+Apoe/ mice received a chow diet for 14 weeks. Analysis of the gene expression of Hdac9 (F) in different tissues normalized to Hprt1. n = 4–9 mice per genotype. Two-way ANOVA with Sidak’s multiple comparisons test. (G) Measurement of mRNA expression of pro- inflammatory cytokines in atherosclerotic aortas of Hdac9DCREApoe/ and control Hdac9+/+Apoe/ mice. n = 6 mice per group. See also Figures S2A–S2K. (H–J) Hdac9DCREApoe/ and control Hdac9+/+Apoe/ mice received a chow diet for 28 weeks. Cytokines and chemokines were measured in plasma by the Luminex multiplex cytokine detection platform. n = 6–10 mice per genotype. Two-sided Mann-Whitney test (Il-1b and Il-18) and two-sided unpaired t test (Il-6). See also Figures S2L–S2T. Data are mean ± SEM.

Article Snippet: MST-Red-NLRP3 was used at a fixed concentration of 30 nM and mixed with equal volumes of serial dilutions of recombinant human HDAC9 (OriGene Technologies, Inc, Rockville, USA), resulting in a final NLRP3 concentration of 15 nM.

Techniques: Control, Staining, Immunostaining, Gene Expression, Expressing, Clinical Proteomics, Luminex, Multiplex Assay, MANN-WHITNEY

Journal: Immunity

Article Title: A cis-regulatory element controls expression of histone deacetylase 9 to fine-tune inflammasome-dependent chronic inflammation in atherosclerosis.

doi: 10.1016/j.immuni.2025.01.003

Figure Lengend Snippet: Figure 2. Deletion of the CRE upregulates Hdac9 expression in myeloid cells to aggravate atherosclerosis (A–C) Analyses of Hdac9 expression in atherosclerosis-relevant cell types isolated from spleen (myeloid cells), and bone marrow (BMDMs) of Hdac9DCREApoe/

Article Snippet: MST-Red-NLRP3 was used at a fixed concentration of 30 nM and mixed with equal volumes of serial dilutions of recombinant human HDAC9 (OriGene Technologies, Inc, Rockville, USA), resulting in a final NLRP3 concentration of 15 nM.

Techniques: Expressing, Isolation

Journal: Immunity

Article Title: A cis-regulatory element controls expression of histone deacetylase 9 to fine-tune inflammasome-dependent chronic inflammation in atherosclerosis.

doi: 10.1016/j.immuni.2025.01.003

Figure Lengend Snippet: Figure 3. The CRE controls Hdac9 expression to promote inflammasome activation (A–C) Hdac9DCREApoe/ (Hdac9DCRE) and control Hdac9+/+Apoe/ (Hdac9+/+) BMDMs were stimulated with LPS (300 ng/mL) for 3 h and nigericin (5 mM) for 45 min. (A) Quantification of caspase-1 activity using a bioluminescent Caspase-Glo 1 inflammasome assay kit. RLU indicates relative luciferase units. n = 5 mice per genotype. Two-sided unpaired t test. (B) Representative immunoblot depicting caspase-1 cleavage. Indicated on the left of each membrane is the molecular weight marker in kDa. (C) Quantification of cleaved caspase-1 normalized to actin. n = 5 independent experiments. Two-sided unpaired t test. See also Figures S5A–S5H. (D–J) Genetic and pharmacological inhibition of Hdac9. Hdac9/Apoe/ (Hdac9/), control Hdac9+/+Apoe/ (Hdac9+/+), and TMP195-treated Apoe/

Article Snippet: MST-Red-NLRP3 was used at a fixed concentration of 30 nM and mixed with equal volumes of serial dilutions of recombinant human HDAC9 (OriGene Technologies, Inc, Rockville, USA), resulting in a final NLRP3 concentration of 15 nM.

Techniques: Expressing, Activation Assay, Control, Activity Assay, Luciferase, Western Blot, Membrane, Molecular Weight, Marker, Inhibition

Journal: Immunity

Article Title: A cis-regulatory element controls expression of histone deacetylase 9 to fine-tune inflammasome-dependent chronic inflammation in atherosclerosis.

doi: 10.1016/j.immuni.2025.01.003

Figure Lengend Snippet: Figure 4. HDAC9 binds and mediates deacetylation of NLRP3 for inflammasome activation (A) HEK293 cells were transiently co-transfected with V5-HDAC9 or V5-empty control vector and FLAG-tagged full-length NLRP3, NLRC4, or AIM2. Shown are representative immunoblots depicting the binding of HDAC9 to NLRP3, but not NLRC4, and AIM2. n = 4 independent experiments. See also Figures S5O–S5Q. (B) HEK293 cells were transiently co-transfected with V5-HDAC9 and FLAG-tagged full-length NLRP3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showing interactors of NLRP3. n = 4 independent experiments. (C and D) Representative immunoblots depicting the binding of HDAC9 to specific domains of NLRP3 (all FLAG-tagged). n = 3–4 independent experiments. Shown below (C) is a cartoon depicting NLRP3 domains. (E) THP1 macrophages were challenged with LPS (200 ng/mL) for 6 h, followed by stimulation with 5 mM of nigericin for 60 min. Endogenous NLRP3 (bottom) was immunoprecipitated with an anti-NLRP3 antibody and endogenous HDAC9 (upper) was revealed by an anti-HDAC9 antibody. Shown are representative im- munoblots depicting the binding of HDAC9 to NLRP3 at endogenous level. n = 3 independent experiments. Bands for HDAC9 and NLRP3 as well as IgGH and IgGL (as immunoprecipitation [IP] side products) are indicated. (F) Protein-protein interactions between HDAC9 and NLRP3 were analyzed in solution applying MST. 15 nM MST-red-NLRP3 was titrated against increasing concentrations of HDAC9. Plotted is the fraction of bound MST-red-NLRP3 (fraction bound) over the indicated concentrations of HDAC9 (log scale). Data are mean ± SD, with 4 data points for each concentration of HDAC9. Two independent titrations measured in two separate sets of capillaries. (G–K) HEK293 cells were transiently co-transfected with V5-HDAC9 and FLAG-tagged full-length or mutant NLRP3. (G) Representative immunoblot of acetylation of NLRP3. (H) Quantification of acetylated NLRP3 normalized to actin. n = 10 independent experiments. Two-sided unpaired t test. (I) Quantification of acetylated NLRP3-DPYD normalized to actin. Representative immunoblots are shown in Figure S5R. n = 5 independent experiments. Two-sided unpaired t test. See also Figure S5R. (J) Representative immunoblot showing NLRP3 oligomerization in transfected HEK293 cells stimulated with nigericin and analyzed by blue native PAGE. (K) Quantification of NLRP3 oligomers normalized to actin. n = 7 independent experiments. Two-sided unpaired t test. Data are mean ± SEM.

Article Snippet: MST-Red-NLRP3 was used at a fixed concentration of 30 nM and mixed with equal volumes of serial dilutions of recombinant human HDAC9 (OriGene Technologies, Inc, Rockville, USA), resulting in a final NLRP3 concentration of 15 nM.

Techniques: Activation Assay, Transfection, Control, Plasmid Preparation, Western Blot, Binding Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Protein-Protein interactions, Concentration Assay, Mutagenesis, Blue Native PAGE

Journal: Immunity

Article Title: A cis-regulatory element controls expression of histone deacetylase 9 to fine-tune inflammasome-dependent chronic inflammation in atherosclerosis.

doi: 10.1016/j.immuni.2025.01.003

Figure Lengend Snippet: Figure 5. HDAC9-targeted immunotherapy stabilizes atherosclerotic plaques (A–D) Apoe/ mice receiving Western-type diet for 8 weeks were treated with TMP195-NB (10 mg/kg) or placebo twice a week starting from the 5th week of diet, when plaques have already developed. (A) Experimental outline. (B) Representative Masson trichrome staining. Dashed lines and asterisks indicate necrotic core areas. (C) Quantification of necrotic core area. n = 9–11 mice per genotype. Two-sided unpaired t test. (D) Vulnerable plaque index. n = 9–11 mice per genotype. Two-sided unpaired t test. See also Figures S7A–S7L. (E–H) Carotid atherosclerotic plaque specimens from patients who underwent endarterectomy for treatment of symptomatic or asymptomatic carotid stenosis were analyzed. (E) Experimental outline. (F) Associations of plaque HDAC9 expression (1 SD increment) with large lipid core (binary traits) as derived from logistic regression analyses adjusted for age, sex, and vascular risk. Shown are odds ratios (ORs) and the 95% confidence interval. N = 615 individuals (age and sex adjusted) and N = 530 individuals (age, sex, and vascular risk factors adjusted). See also Figures S8A–S8C. (G) Plaque HDAC9 expression (inverse-rank transformed) in study participants across vulnerable plaque index scores (p value derived from Kruskal-Wallis test). Shown are the median values (central line), the upper and lower quartiles (box limits) and the 1.53 interquartile range (whiskers). 12 outlier observations with HDAC9 expression levels > 100 are not displayed for graphical reasons. Statistics reported with raw data; associations without outliers show a p value = 0.023. (H) Plaque HDAC9 expression (inverse-rank trans- formed) in patients with symptomatic vs. asymptomatic plaques (p value derived from Mann-Whitney U test). (I–L) Monocytes were freshly isolated from patients with advanced atherosclerosis (n = 6) and treated ex vivo with TMP195 or vehicle for 1 h prior to stimulation with LPS (100 ng/mL) for 3 h and nigericin (5 mM) for 60 min. (I) Representative immunoblot of caspase-1 cleavage. (J) Quantification of cleaved caspase-1 normalized to actin. n = 6 independent experiments. Two-sided unpaired t test. Quantification of IL-1b (K) and IL-18 (L) release. n = 6 independent experi- ments. Two-sided unpaired t test. See also Figures S8D and S8E. Data are mean ± SEM.

Article Snippet: MST-Red-NLRP3 was used at a fixed concentration of 30 nM and mixed with equal volumes of serial dilutions of recombinant human HDAC9 (OriGene Technologies, Inc, Rockville, USA), resulting in a final NLRP3 concentration of 15 nM.

Techniques: Western Blot, Staining, Expressing, Derivative Assay, Transformation Assay, MANN-WHITNEY, Isolation, Ex Vivo

Journal: Journal of Cellular and Molecular Medicine

Article Title: Indole‐3‐Carboxaldehyde Inhibits Inflammatory Response and Lipid Accumulation in Macrophages Through the miR ‐1271‐5p/ HDAC9 Pathway

doi: 10.1111/jcmm.70263

Figure Lengend Snippet: ICA reduces the stability of HDAC9 mRNA. (A) 293 T cells transfected with pGL3‐HDAC9 or pGL3‐NC were treated with ICA (50 μM) or DMSO for 24 h. Cell lysates were used to detect the luciferase activity. (B) The half‐life of HDAC9 mRNA in THP‐1 macrophage‐derived foam cells pretreated with ICA (50 μM) or DMSO for 24 h were determined with the addition of ActD. (C) 293 T cells transfected with pGL3‐HDAC9 3′‐UTR or pGL3‐NC were incubated with ICA (50 μM) or DMSO. The luciferase activity was then examined after 24 h. Data represent the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01. NS indicates not significant.

Article Snippet: Briefly, the luciferase reporter plasmids containing human HDAC9 promoter region of 2000 bp (pGL3‐HDAC9) were constructed by Genechem (Shanghai, China), with the empty pGL3 vector as a negative control (pGL3‐NC).

Techniques: Transfection, Luciferase, Activity Assay, Derivative Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Indole‐3‐Carboxaldehyde Inhibits Inflammatory Response and Lipid Accumulation in Macrophages Through the miR ‐1271‐5p/ HDAC9 Pathway

doi: 10.1111/jcmm.70263

Figure Lengend Snippet: Identification of HDAC9 as a direct target of miR‐1271‐5p. (A) After treatment of THP‐1 macrophage‐derived foam cells with ICA (50 μM) or DMSO for 24 h, qRT‐PCR was employed to analyse the expression of miR‐182‐5p, miR‐383‐5p and miR‐27a‐3p. (B) The binding site between miR‐1271‐5p and HDAC9 3′‐UTR and corresponding mutation. (C) Free energy score for the interaction of miR‐1271‐5p with HDAC9 3′‐UTR. (D) The luciferase activity was detected in 293 T cells transfected with HDAC9‐WT or HDAC9‐Mut plasmids plus 50 nM of miR‐1271‐5p mimic or mimic control for 48 h. (E, F) THP‐1 macrophage‐derived foam cells were transfected with miR‐1271‐5p mimic/inhibitor or their negative controls (50 nM) for 48 h. (E) QRT‐PCR analysis of miR‐1271‐5p expression. (F) Detection of HDAC9 expression using qRT‐PCR and western blot. Data are presented as mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001. NS indicates not significant.

Article Snippet: Briefly, the luciferase reporter plasmids containing human HDAC9 promoter region of 2000 bp (pGL3‐HDAC9) were constructed by Genechem (Shanghai, China), with the empty pGL3 vector as a negative control (pGL3‐NC).

Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Transfection, Control, Western Blot

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Decreased expression of HDAC9 in degenerated IVD in patients. A , B GSE56081 dataset (NP tissues from patients with IVDD and normal controls ) were analyzed by gene set enrichment analysis (GSEA) based on the Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. C Raincloud plot exhibited DEGs related to protein modification such as ubiquitination, deubiquitination, phosphorylation, dephosphorylation, acylation, and diacylation based on the Gene Ontology resource knowledge base (Raincloud plot was drawn using https://www.chiplot.online/ ). D Heatmap presented the top three upregulated and downregulated DEGs from C . E UpSet plot displayed the intersections between six deacetylation genes and DEGs from the GSE56081 dataset

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Expressing, Modification, De-Phosphorylation Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: HDAC9 is decreased in degenerated IVD in aged mice. A MRI scan of the lumbar spine and intervertebral discs (IVDs) of 6-month and 18-month-old mice. B Coronal sections of disc compartments stained by safranin-O/fast green. Scale bar: black, 500 μm; yellow, 200 μm. C Fluorescence staining of HDAC9 in disc compartments. Scale bar: White, 100 μm; Yellow, 50 μm. Quantification of staining for HDAC9-positive cells. Data are represented as mean ± SD ( n = 6). A p -value of less than 0.05 was considered significant using an unpaired student’s t -test. NS: not significant

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Staining, Fluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Deletion of HDAC9 contributes to degeneration of IVD during aging. A Schematic diagram of the HDAC9 knockout design showing partial mouse HDAC9 gene (top), sequences of two gRNAs used for deletion of exon 3 to 6 (middle), and the truncated DNA was confirmed by sequencing validation (bottom). B MRI scan of the lumbar spine and IVDs of HDAC9 WT mice and HDAC9 KO mice at 1, 3, and 6 months. C , D Coronal sections of disc compartments stained and analyzed by safranin-O/fast green. Scale bar: black, 500 μm; yellow, 200 μm. Quantification of area ratios of NP cell band/IVD ( E ) and NP/IVD ( F ). G Body weight of WT mice and HDAC9 KO mice at 1, 3, and 6 months. H Fluorescence staining of HDAC9 in NP compartment. Scale bar: 25 μm. I Apoptotic cells in the NP compartment were detected by TUNEL staining. Scale bar: 25 μm. Data are represented as mean ± SD ( n = 6). A p value of less than 0.05 was considered significant using an unpaired Student’s t -test. NS, not significant

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Knock-Out, Sequencing, Staining, Fluorescence, TUNEL Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: mRNA-seq analysis determines HDAC9-regulated genes in NP cells. A Schematic of the NP cells isolated from HDAC9 KO and HDAC9 WT mice ( n = 3) were subject to mRNA-seq. B Volcano plot of expression of genes. Green, downregulated DEGs; red, upregulated DEGs; blue, not significant. C Heatmap of the expression profiling of the top 20 upregulated and downregulated DEGs. The red color and green color represent upregulated DEGs and downregulated DEGs, respectively. D Schematic of GSEA enrichment analysis of genes from mRNA-seq. GSEA plot depicting the enrichment of genes in deubiquitinase activity, PI3K-Akt signaling pathway, muscle cell proliferation, deacetylase activity, cytokine–cytokine receptor interaction, oxidative phosphorylation, histone acetyltransferase activity, glycolysis/gluconeogenesis pathway, and modification-dependent protein binding

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Isolation, Expressing, Activity Assay, Histone Deacetylase Assay, Modification, Protein Binding

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Co-IP/MS analysis determines HDAC9-interacting proteins in NP cells. A The NP cells isolated from C57BL/6J mice were subjected to co-IP/MS using anti-HDAC9 and IgG. B , C The protein identification results from database screening and the percentage of specific HDAC9-interacting proteins in NP cells. D The number of HDAC9-interacting proteins possibly modified by acetylation (611), phosphorylation (705), and ubiquitination (893) shown according to the PhosphoSitePlus database. E Eukaryotic Ortholog Groups (KOG) of proteins annotation of HDAC9-interacting proteins. F The number of HDAC9-interacting proteins that function as transcription factors in mice (36) and humans (80). G Lollipop plot presented the 34 common transcription factors in the classified 16 transcription factor families (Lollipop plot was drawn using https://www.chiplot.online/ ). H , I GO annotations determined the molecular functions of the HDAC9-interacting proteins

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Co-Immunoprecipitation Assay, Isolation, Modification

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Deficiency of HDAC9 affects cell apoptosis, viability, and regulates RUNX3 in NP tissue. A , B Fluorescence staining and quantification of percentage of Cyclin D1 positive cells, p21 positive cells, p53 positive cells, and RUNX3 positive cells in the NP tissues of 6-month-old HDAC9 WT mice and HDAC9 KO mice. Scale bar, 25 μm. C The disc tissues were immunoprecipitated with anti-RUNX3 and acetylation of RUNX3 was analyzed using an anti-acetyl-lysine. Data are represented as mean ± SD ( n = 6). A p value of less than 0.05 was considered significant using an unpaired Student’s t -test. NS, not significant

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Fluorescence, Staining, Immunoprecipitation

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: HDAC9 knockdown increases cell apoptosis and inhibits cell viability of NP cells in vitro. A Identification of isolated NP cells by collagen II using fluorescence staining. Scale bar, 100 μm. B , C NP cells were infected with lentivirus-mediated shHDAC9 (Lt.shHDAC9-1, 2, and 3) for 72 h. The relative mRNA level and protein level of HDAC9 in Lt.shHDAC9-infected NP cells were detected by RT–qPCR and western blot. D Cell viability of NP cells was measured by CCK-8 assay. E Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. The relative mRNA level of p21 ( F ), cyclin D1 ( G ), and PUMA ( H ). I , J The protein level of p21, cyclin D1, p53, and PUMA. K HDAC9 KO NP cells were infected with adenovirus containing cDNA of HDAC9 (Ad.HDAC9) for 72 h and the protein level of HDAC9 was detected. L Cell viability of NP cells was measured by CCK-8 assay. M Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. Data are represented as mean ± SD ( n = 3). A p -value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: In Vitro, Isolation, Fluorescence, Staining, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Knockdown of HDAC9 increases acetylation and inhibits ubiquitin–proteasomal degradation of RUNX3. A , B The mRNA and protein level of RUNX3 in Lt.shHDAC9-infected NP cells. C Double fluorescence staining of HDAC9 and RUNX3 in isolated NP cells. Scale bar, 50 μm. D NP cells were immunoprecipitated with anti-RUNX3 to analyze of the interaction of HDAC9 and RUNX3. E The acetylation of RUNX3 in HDAC9 knockdown cells. F Lt.shHDAC9-infected cells were treated with MG132 for 8 h and immunoprecipitated with anti-RUNX3 to detect ubiquitination of RUNX3 using anti-ubiquitin (ubi) antibody. G Lt.shHDAC9-infected cells were treated with cycloheximide (CHX) for indicated time points, and then RUNX3 remaining protein level was detected by western blot. H NP cells were infected with Lt.shHDAC9 and Lt.shRUNX3 for 72 h and the protein level of RUNX3 was detected. I Cell viability of NP cells was measured by CCK-8 assay. J Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. K The protein level of p53, p21, PUMA and Cyclin D1 were detected by western blot. Data are represented as mean ± SD ( n = 3). A p value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Infection, Fluorescence, Staining, Isolation, Immunoprecipitation, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Overexpression of HDAC9 alleviates surgery-induced IVDD in mice. A Schematic of HDAC9 overexpression and animal working model. Surgically induced IVDD model was induced by puncturing at the L5/6 IVD with a 30-gauge needle, and adenovirus carrying Col2a1-promoter mediated overexpression of HDAC9 (HDAC9 Col2a1 ) and NC Col2a1 were injected to the NP tissue during needle puncture. After 4 weeks of surgery, the mice were sacrificed. B MRI scan of the lumbar spine. C , D Coronal sections of disc compartments stained and analyzed by safranin-O/fast green. Scale bar: black, 500 μm; yellow, 200 μm. E , F Apoptotic cells in the NP compartment were detected by TUNEL staining. Scale bar, 25 μm. G , H Fluorescence staining of HDAC9 in NP compartment. Scale bar, 25 μm. I The acetylation level of RUNX3 in disc tissues. Data are represented as mean ± SD ( n = 6). A p value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: The primary antibodies used were anti-HDAC9 (1:100, ab109446), anti-RUNX3 (1:50, Abcam, ab135248), anti-p21 (1:100, Proteintech, Wuhan, China, 10,355–1-AP), anti-p53 (1:100, Proteintech, 60,283–2-Ig), anti-cyclin D1 (1:100, Proteintech, 26,939–1-AP), and anti-collagen II (1:100, Affinity, AF0135) at 4 °C overnight.

Techniques: Over Expression, Injection, Staining, TUNEL Assay, Fluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Decreased expression of HDAC9 in degenerated IVD in patients. A , B GSE56081 dataset (NP tissues from patients with IVDD and normal controls ) were analyzed by gene set enrichment analysis (GSEA) based on the Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. C Raincloud plot exhibited DEGs related to protein modification such as ubiquitination, deubiquitination, phosphorylation, dephosphorylation, acylation, and diacylation based on the Gene Ontology resource knowledge base (Raincloud plot was drawn using https://www.chiplot.online/ ). D Heatmap presented the top three upregulated and downregulated DEGs from C . E UpSet plot displayed the intersections between six deacetylation genes and DEGs from the GSE56081 dataset

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Expressing, Modification, Ubiquitin Proteomics, Phospho-proteomics, De-Phosphorylation Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: HDAC9 is decreased in degenerated IVD in aged mice. A MRI scan of the lumbar spine and intervertebral discs (IVDs) of 6-month and 18-month-old mice. B Coronal sections of disc compartments stained by safranin-O/fast green. Scale bar: black, 500 μm; yellow, 200 μm. C Fluorescence staining of HDAC9 in disc compartments. Scale bar: White, 100 μm; Yellow, 50 μm. Quantification of staining for HDAC9-positive cells. Data are represented as mean ± SD ( n = 6). A p -value of less than 0.05 was considered significant using an unpaired student’s t -test. NS: not significant

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Staining, Fluorescence

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Deletion of HDAC9 contributes to degeneration of IVD during aging. A Schematic diagram of the HDAC9 knockout design showing partial mouse HDAC9 gene (top), sequences of two gRNAs used for deletion of exon 3 to 6 (middle), and the truncated DNA was confirmed by sequencing validation (bottom). B MRI scan of the lumbar spine and IVDs of HDAC9 WT mice and HDAC9 KO mice at 1, 3, and 6 months. C , D Coronal sections of disc compartments stained and analyzed by safranin-O/fast green. Scale bar: black, 500 μm; yellow, 200 μm. Quantification of area ratios of NP cell band/IVD ( E ) and NP/IVD ( F ). G Body weight of WT mice and HDAC9 KO mice at 1, 3, and 6 months. H Fluorescence staining of HDAC9 in NP compartment. Scale bar: 25 μm. I Apoptotic cells in the NP compartment were detected by TUNEL staining. Scale bar: 25 μm. Data are represented as mean ± SD ( n = 6). A p value of less than 0.05 was considered significant using an unpaired Student’s t -test. NS, not significant

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Knock-Out, Sequencing, Biomarker Discovery, Staining, Fluorescence, TUNEL Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: mRNA-seq analysis determines HDAC9-regulated genes in NP cells. A Schematic of the NP cells isolated from HDAC9 KO and HDAC9 WT mice ( n = 3) were subject to mRNA-seq. B Volcano plot of expression of genes. Green, downregulated DEGs; red, upregulated DEGs; blue, not significant. C Heatmap of the expression profiling of the top 20 upregulated and downregulated DEGs. The red color and green color represent upregulated DEGs and downregulated DEGs, respectively. D Schematic of GSEA enrichment analysis of genes from mRNA-seq. GSEA plot depicting the enrichment of genes in deubiquitinase activity, PI3K-Akt signaling pathway, muscle cell proliferation, deacetylase activity, cytokine–cytokine receptor interaction, oxidative phosphorylation, histone acetyltransferase activity, glycolysis/gluconeogenesis pathway, and modification-dependent protein binding

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Isolation, Expressing, Activity Assay, Histone Deacetylase Assay, Phospho-proteomics, Modification, Protein Binding

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Co-IP/MS analysis determines HDAC9-interacting proteins in NP cells. A The NP cells isolated from C57BL/6J mice were subjected to co-IP/MS using anti-HDAC9 and IgG. B , C The protein identification results from database screening and the percentage of specific HDAC9-interacting proteins in NP cells. D The number of HDAC9-interacting proteins possibly modified by acetylation (611), phosphorylation (705), and ubiquitination (893) shown according to the PhosphoSitePlus database. E Eukaryotic Ortholog Groups (KOG) of proteins annotation of HDAC9-interacting proteins. F The number of HDAC9-interacting proteins that function as transcription factors in mice (36) and humans (80). G Lollipop plot presented the 34 common transcription factors in the classified 16 transcription factor families (Lollipop plot was drawn using https://www.chiplot.online/ ). H , I GO annotations determined the molecular functions of the HDAC9-interacting proteins

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Co-Immunoprecipitation Assay, Isolation, Modification, Phospho-proteomics, Ubiquitin Proteomics

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Deficiency of HDAC9 affects cell apoptosis, viability, and regulates RUNX3 in NP tissue. A , B Fluorescence staining and quantification of percentage of Cyclin D1 positive cells, p21 positive cells, p53 positive cells, and RUNX3 positive cells in the NP tissues of 6-month-old HDAC9 WT mice and HDAC9 KO mice. Scale bar, 25 μm. C The disc tissues were immunoprecipitated with anti-RUNX3 and acetylation of RUNX3 was analyzed using an anti-acetyl-lysine. Data are represented as mean ± SD ( n = 6). A p value of less than 0.05 was considered significant using an unpaired Student’s t -test. NS, not significant

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Fluorescence, Staining, Immunoprecipitation

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: HDAC9 knockdown increases cell apoptosis and inhibits cell viability of NP cells in vitro. A Identification of isolated NP cells by collagen II using fluorescence staining. Scale bar, 100 μm. B , C NP cells were infected with lentivirus-mediated shHDAC9 (Lt.shHDAC9-1, 2, and 3) for 72 h. The relative mRNA level and protein level of HDAC9 in Lt.shHDAC9-infected NP cells were detected by RT–qPCR and western blot. D Cell viability of NP cells was measured by CCK-8 assay. E Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. The relative mRNA level of p21 ( F ), cyclin D1 ( G ), and PUMA ( H ). I , J The protein level of p21, cyclin D1, p53, and PUMA. K HDAC9 KO NP cells were infected with adenovirus containing cDNA of HDAC9 (Ad.HDAC9) for 72 h and the protein level of HDAC9 was detected. L Cell viability of NP cells was measured by CCK-8 assay. M Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. Data are represented as mean ± SD ( n = 3). A p -value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Knockdown, In Vitro, Isolation, Fluorescence, Staining, Infection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Flow Cytometry, Comparison

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Knockdown of HDAC9 increases acetylation and inhibits ubiquitin–proteasomal degradation of RUNX3. A , B The mRNA and protein level of RUNX3 in Lt.shHDAC9-infected NP cells. C Double fluorescence staining of HDAC9 and RUNX3 in isolated NP cells. Scale bar, 50 μm. D NP cells were immunoprecipitated with anti-RUNX3 to analyze of the interaction of HDAC9 and RUNX3. E The acetylation of RUNX3 in HDAC9 knockdown cells. F Lt.shHDAC9-infected cells were treated with MG132 for 8 h and immunoprecipitated with anti-RUNX3 to detect ubiquitination of RUNX3 using anti-ubiquitin (ubi) antibody. G Lt.shHDAC9-infected cells were treated with cycloheximide (CHX) for indicated time points, and then RUNX3 remaining protein level was detected by western blot. H NP cells were infected with Lt.shHDAC9 and Lt.shRUNX3 for 72 h and the protein level of RUNX3 was detected. I Cell viability of NP cells was measured by CCK-8 assay. J Apoptotic cells were stained with annexin V/propidium iodide and quantified by flow cytometry. K The protein level of p53, p21, PUMA and Cyclin D1 were detected by western blot. Data are represented as mean ± SD ( n = 3). A p value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Knockdown, Ubiquitin Proteomics, Infection, Fluorescence, Staining, Isolation, Immunoprecipitation, Western Blot, CCK-8 Assay, Flow Cytometry, Comparison

Journal: Cellular & Molecular Biology Letters

Article Title: Molecular mechanism and therapeutic potential of HDAC9 in intervertebral disc degeneration

doi: 10.1186/s11658-023-00517-x

Figure Lengend Snippet: Overexpression of HDAC9 alleviates surgery-induced IVDD in mice. A Schematic of HDAC9 overexpression and animal working model. Surgically induced IVDD model was induced by puncturing at the L5/6 IVD with a 30-gauge needle, and adenovirus carrying Col2a1-promoter mediated overexpression of HDAC9 (HDAC9 Col2a1 ) and NC Col2a1 were injected to the NP tissue during needle puncture. After 4 weeks of surgery, the mice were sacrificed. B MRI scan of the lumbar spine. C , D Coronal sections of disc compartments stained and analyzed by safranin-O/fast green. Scale bar: black, 500 μm; yellow, 200 μm. E , F Apoptotic cells in the NP compartment were detected by TUNEL staining. Scale bar, 25 μm. G , H Fluorescence staining of HDAC9 in NP compartment. Scale bar, 25 μm. I The acetylation level of RUNX3 in disc tissues. Data are represented as mean ± SD ( n = 6). A p value of less than 0.05 was considered significant using one-way ANOVA and Tukey’s multiple comparison test

Article Snippet: Subsequently, the precipitated proteins were incubated with an anti-HDAC9 antibody (1:5000, Abcam, Cambridge, UK, ab109446), anti-RUNX3 antibody, anti-acetyl lysine antibody (1:1000, Abcam, ab190479), or anti-ubiquitin (Ubi) antibody (1:5000, Abcam, ab134953).

Techniques: Over Expression, Injection, Staining, TUNEL Assay, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Augmentation of a neuroprotective myeloid state by hematopoietic cell transplantation

doi: 10.1101/2023.03.10.532123

Figure Lengend Snippet: ( a ) Immunofluorescent stain for Gpnmb in the spinal cord white matter. Scale bar is 20µm. ( b ) Immunofluorescence images of the grey/white matter border in the spinal cord. Neuronal nuclei show bright Histone deacetylase 9 (Hdac9) signal. Light blue arrows indicate colocalization of myeloid cells and Hdac9 positive nuclei. Scale bar is 30µm.

Article Snippet: The following primary antibodies were used for immunofluorescence staining: C4 (abcam, ab11863), CD206 (R&D systems, AF2535), GFAP (Invitrogen, 13-0300), GFP (abcam, ab13970), GPNMB (Bioss, BS- 2684R), HDAC9 (abcam, ab109446), Iba1 (FUJIFILM Wako, 019-19741), Iba1 (abcam, ab5076), IQGAP1 (abcam, ab133490), Olig2 (Millipore Sigma, AB9610), Spp1 (Santa Cruz Biotechnology, sc-21742), Stat1 (Cell Signaling, 14994).

Techniques: Staining, Immunofluorescence, Histone Deacetylase Assay