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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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: To determine whether HDAC9 regulates RUNX3 protein stability, Lt.shHDAC9-infected cells were treated with protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml, MCE, USA, HY-12320) and the protein expression of RUNX3 was measured at 0, 4, 8, and 12 h post-CHX treatment.

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

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A,B) HUVEC were exposed to hypoxia (4 h) or hypoxia followed by reoxygenation (1-24 h) or remained untreated. (A) Cezanne transcript levels were quantified by real-time PCR. Data were pooled from 3 independent experiments. (B) Cytosolic lysates were tested by Western blotting using anti-Cezanne antibodies and by using anti-α-tubulin antibodies to assess total protein levels. Representative blots (upper panels) and results from densitometry analysis of five experiments (lower panel) are shown. (C) HDMEC or HCMEC were exposed to hypoxia (4 h) or remained untreated. Cezanne transcript levels were quantified by real-time PCR. Data were pooled from 3 independent experiments. (D) HUVEC or HCMEC were exposed to 1%, 2%, 5% or normoxia (21%) for 4 h. Cezanne transcript levels were quantified by real-time PCR. Data were pooled from 3 independent experiments. (E,F) HUVEC were exposed to CT8730 (1 μM) or SB202190 (50 μM) for 1 h or were treated with vehicle alone and then exposed to hypoxia (4 h) or remained untreated. (E) Cezanne transcript levels were quantified by real-time PCR. (F) Cytosolic lysates were tested by Western blotting using anti-Cezanne antibodies and by using anti-α-tubulin antibodies to assess total protein levels. n.s., non-specific band. (G) HUVEC were treated with p38α-specific siRNA (si-p38) or with a scrambled, non-targeting sequence and were then exposed to hypoxia (4 h) or hypoxia (4 h) followed by reoxygenation (4 h) or remained untreated (normoxia). Cezanne transcript levels were quantified by real-time PCR.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Sequencing

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A) HUVEC or HCMEC were exposed to hypoxia for 15 min-4 h or remained untreated (normoxia). Some cultures were then reoxygenated for 1 h. Cytosolic lysates were tested by Western blotting using anti-phosphorylated ATF2 antibodies or by using anti-α-tubulin antibodies to assess total protein levels. Data are representative of three independent experiments. (B) HUVEC were treated with siRNA sequences that target Cezanne (Cez 1, Dharmacon; Cez 2, SMARTpool), ATF2 (ATF2 1, Dharmacon; ATF2 2, SMARTpool) or SHP2, or with scrambled, non-targeting sequences as a control (Scr). They were then exposed to hypoxia (4 h) or remained untreated (normoxia). Levels of Cezanne or ATF2 were quantified by real-time PCR. Data were pooled from 3 independent experiments.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

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

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: HUVEC were treated with Cezanne-specific siRNA (si-Cezanne from Dharmacon) or with a scrambled, non-targeting sequence and were then exposed to hypoxia (4 h) or hypoxia (4 h) followed by reoxygenation (4-20 h) or remained untreated. (A) Levels of E-selectin, VCAM- 1 or ICAM-1 transcripts were quantified by real-time PCR. Data were pooled from three independent experiments. (B) Cytosolic or nuclear lysates were tested by Western blotting using anti-IκBα, anti-RelA or anti-Ser536 phosphorylated RelA antibodies and by using anti-α-tubulin or anti-Lamin B antibodies to assess total protein levels. Data are representative of three independent experiments.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: HUVEC were treated with Cezanne-specific siRNA (si-Cezanne from Dharmacon) or with a scrambled, non-targeting sequence and were then exposed to hypoxia (4 h) or hypoxia (4 h) followed by reoxygenation (2-20 h) or remained untreated. (A) TRAF6 immunoprecipitates or lysates were tested by Western blotting using anti-TRAF6 antibodies. (B) TRAF6 immunoprecipitates were tested by Western blotting using antibodies that recognise Lys63-polyubiquitin. n.s., non-specific. Data are representative of three independent experiments that gave closely similar results.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Sequencing, Western Blot

Journal: Circulation research

Article Title: Cezanne regulates inflammatory responses to hypoxia in endothelial cells by targeting TRAF6 for deubiquitination

doi: 10.1161/CIRCRESAHA.111.300119

Figure Lengend Snippet: (A,B) Expression levels of Cezanne were assessed in left kidneys of male Fisher rats (n=4) that were exposed to ischemia followed by reperfusion. Basal levels of Cezanne were measured in the uninjured right kidney of each animal. (A) Cezanne transcript levels were quantified in kidneys exposed to ischemia-reperfusion (6 h) and uninjured kidneys by real-time PCR and normalised by quantifying β-actin transcripts. (B) Lysates from kidneys exposed to ischemia-reperfusion (6 h) or uninjured kidneys were tested by Western blotting using anti-Cezanne antibodies or by using anti-α-tubulin antibodies to assess total protein levels. A representative blot and results from densitometry analysis are shown. (C,D) Left kidneys of wild-type or CezanneGT/GT mice (n=6 per group) were exposed to ischemia followed by reperfusion for 6 h whereas contralateral kidneys were uninjured. (C) Cezanne transcript levels were quantified in kidneys exposed to ischemia-reperfusion and uninjured kidneys by real-time PCR and normalised by quantifying β-actin transcripts. (D) Cezanne expression was assessed by immunohistochemistry in kidneys exposed to ischemia-reperfusion. Positive staining was observed in wild-type tissues in vascular and glomerular EC (arrows), in podocytes (arrowhead) and epithelial cells.

Article Snippet: Reagents and antibodies Anti-Cezanne (Proteintech Europe Ltd), Anti-RelA (p65), anti-κBα, anti-TRAF6, anti-Lamin B (Santa Cruz Biotechnology), phosphorylated anti-ATF2 (Thr71), phosphorylated anti-RelA (Ser536) (Cell Signalling Technology), anti-ubiquitin (Invitrogen), anti- Lys63 polyubiquitin, anti-GAPDH (Merck-Millipore), anti-α-tubulin (Sigma-Aldrich) anti-kidney injury marker-1 (R&D Systems) and polyclonal goat anti-rabbit and anti-mouse conjugated horse radish peroxidise (HRP) (Dako) antibodies were obtained commercially.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Staining