recombinant human hdac5  (BPS Bioscience)

Journal: bioRxiv

Article Title: HDAC5 -encoded Microprotein NISM Mediates Nucleolar Formation and Ribosomal RNA Synthesis

doi: 10.64898/2026.02.21.707204

Figure Lengend Snippet: (A-B) BedGraph tracks of HEK293T Ribo-seq coverage across the entire HDAC5 transcript (A) and zoomed in on the NISM smORF within the 5′-UTR (B). In (A), the NISM smORF is highlighted in yellow. HDAC5 is on the negative strand and thus the 5′ to 3′ orientation runs right to left. The top tracks show read coverage for cells pre-treated with harringtonine (Harr) to capture translation initiation sites and the bottom tracks show read coverage for untreated cells lysed in the presence of cycloheximide to capture elongating ribosomes. PhyloCSF scores for each reading frame are shown below the RefSeq transcript tracks. Possible start codons in the NISM smORF are denoted by ‘M’ in (B). (C) Sequence alignment showing amino acid level conservation of NISM across distant mammals. (D) Immunoblot analysis of short and long NISM-ALFA expression in HEK293T cells. (E) ESMFold predicted structure of short NISM. Low confidence predictions are colored yellow and very low confidence predictions are colored orange. (F) Representative immunofluorescence images of NPM1 (red), FBL (cyan), and NISM-ALFA (green) in HEK293T cells transfected with empty vector (EV), short NISM-ALFA, or long NISM-ALFA for 48 h. Nuclei were counter-stained with DAPI (blue). Scale bar, 5 µm. All data are representative of at least two biological replicates.

Article Snippet: Expression of HDAC5 at the protein level was also validated by SDS-PAGE and immunoblot using the HDAC5 rabbit monoclonal antibody (Cell Signaling, 20458) following IP of HDAC5 from 500 μg of total protein from WT or NISM KO U2OS cells.

Techniques: Sequencing, Western Blot, Expressing, Immunofluorescence, Transfection, Plasmid Preparation, Staining

Journal: Biomedicines

Article Title: Secosteroid–2-Pyrazoline Hybrids: Design, Synthesis, Biological Evaluation and Development of Therapeutic Combinations Against ERα-Positive Breast Cancer Cells

doi: 10.3390/biomedicines13123057

Figure Lengend Snippet: Effect of lead secosteroid–2-pyrazoline hybrids 3f , 3j , and 3k on signaling pathways in MCF-7 breast cancer cells. Antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were used for immunoblotting.

Article Snippet: Primary antibodies against androgen receptor (AR), estrogen receptor α (ERα), GREB1, S6K, and its phosphorylated form (p-S6K), Bcl-2, and its phosphorylated form (p-Bcl-2), and α-tubulin were employed (Cell Signaling Technology, Danvers, MA, USA). α-Tubulin served as the internal loading control.

Techniques: Protein-Protein interactions, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic tumor images and tumor weights from KPC Hdac5-WT and KPC Hdac5-KO mouse models treated with vehicle or MRTX1133 (30 mg/kg, i.p., twice daily [bid]) ( n = 5). ( B ) Kaplan-Meier survival curves with log-rank test ( n = 5). ( C and D ) Representative IHC images of tumors in C . IHC scores were quantified in D . Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. ( E ) C57BL/6 mice were orthotopically injected with KPC-Luc cells expressing shNc or shHdac5. Bioluminescence imaging was performed on day 7, followed by treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid). Representative bioluminescence images and corresponding quantification were acquired on day 28 ( n = 5). ( F ) Tumor growth curves in PDX models with treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid) ( n = 5). ( G and H ) Representative IHC images of PDXs and quantified IHC scores ( H ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( A , D – F , and H ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Injection, Expressing, Imaging

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of genes upregulated in KPC Hdac5-KO mice ( n = 5 per genotype; log 2 [fold-change] > 1, P < 0.05, 2,410 genes) identified via RNA-Seq and genes upregulated in shHDAC5-treated PANC-1 cells ( n = 3 per condition; log 2 [fold-change] > 1, P < 0.05, 2,683 genes), revealing a shared subset of 378 genes. ( B ) Bar graph showing the top 10 enriched transcription factors from transcription factor analysis of 378 genes in A using Enrichr. ATF2, activating transcription factor 2. ( C ) Dual-luciferase reporter assays were performed to assess the transcriptional activity of c-Myc in HDAC5-depleted PANC-1 and AsPC-1 cells. Data are presented as mean ± SD ( n = 5). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. ( D ) Heatmap of MYC ChIP-Seq signal intensity (±3 kb around MYC binding sites) in control vs. HDAC5-knockdown PANC-1 cells. ( E ) The average ChIP signal of MYC centered at transcription start site (±3 kb) in indicated groups. ( F ) Venn diagram depicting the overlap between genes with enhanced MYC promoter occupancy after shHDAC5 knockdown, as identified by ChIP-Seq, and genes upregulated in PANC-1 cells following shHDAC5 knockdown, as determined by RNA-Seq. P = 6 × 10 –71 . ( G ) KEGG pathway enrichment analysis of 821 intersecting genes in F . ( H and I ) Western blot analysis of canonical MAPK pathway protein expression under indicated conditions.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: RNA Sequencing, Luciferase, Activity Assay, ChIP-sequencing, Binding Assay, Control, Knockdown, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic images of pancreatic tumors from KPC Hdac5-WT and KPC Hdac5-KO mice after sacrifice. ( B ) Tumor weight analysis in KPC mice treated with vehicle, MRTX1133 (30 mg/kg, i.p. bid), MYCi975 (50 mg/kg, i.p. bid), or their combination ( n = 5). ( C ) Kaplan-Meier survival curves with log-rank test ( n = 5). *** P < 0.001. ( D and E ) Representative IHC images of tumors from treated mice and quantified IHC scores ( E ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent quantifications. ( F and G ) Representative bioluminescence images and quantification ( n = 5). ( H ) Macroscopic images of PDX tumors after 30 days of treatment. ( I and J ) Tumor growth curves ( I ) and final tumor weights ( J ) in PDX models treated with Vehicle + LV-Control, MRTX1133 (30 mg/kg, i.p. bid) + LV-Control, Vehicle + LV-sgMYC (50 μL lentivirus, s.c., weekly), or MRTX1133 + LV-sgMYC. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( B , E , G , I , and J ). *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Scatterplot depicting the antiproliferative effects of a single dose (10 μM) of 1,737 FDA-approved anticancer compounds on PANC-1 and Mia PaCa-2 cells, treated with either short hairpin RNA negative control (shNC) or shHDAC5. ( B ) Heatmap shows the normalized IC 50 ratio of different KRAS G12D inhibitors determined by cell counting kit 8 (CCK8) assay in PANC-1 and AsPC-1 cells ( n = 2). The normalized IC 50 ratio was calculated as the fold-change in IC 50 of shHDAC5 relative to shNC. ( C ) The IC 50 of MRTX1133 was assessed by CCK8 assay in PANC-1 and AsPC-1 cells with HDAC5 knockdown. ( D and E ) Representative images of 3D-cultured HDAC5-depleted cells treated with DMSO or MRTX1133 (PANC-1: 10 μM; AsPC-1: 5 nM; 48 hours). Scale bars = 100 μm. Relative survival of cells ( E ) ( n = 3). ( F ) Cell viability of PANC-1 and AsPC-1 cells expressing shNC or shHDAC5 and treated with DMSO or MRTX1133 (10 μM for PANC-1; 5 nM for AsPC-1), measured by CCK8 assay ( n = 3). ( G ) Representative images and size quantification of PDOs treated with DMSO or MRTX1133 (1 μM) ( n = 5). All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( E – G ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: shRNA, Negative Control, Cell Counting, CCK-8 Assay, Knockdown, Cell Culture, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic tumor images and tumor weights from KPC Hdac5-WT and KPC Hdac5-KO mouse models treated with vehicle or MRTX1133 (30 mg/kg, i.p., twice daily [bid]) ( n = 5). ( B ) Kaplan-Meier survival curves with log-rank test ( n = 5). ( C and D ) Representative IHC images of tumors in C . IHC scores were quantified in D . Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. ( E ) C57BL/6 mice were orthotopically injected with KPC-Luc cells expressing shNc or shHdac5. Bioluminescence imaging was performed on day 7, followed by treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid). Representative bioluminescence images and corresponding quantification were acquired on day 28 ( n = 5). ( F ) Tumor growth curves in PDX models with treatment with vehicle or MRTX1133 (30 mg/kg, i.p., bid) ( n = 5). ( G and H ) Representative IHC images of PDXs and quantified IHC scores ( H ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent IHC quantifications. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( A , D – F , and H ). * P < 0.05, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Injection, Expressing, Imaging

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of genes upregulated in KPC Hdac5-KO mice ( n = 5 per genotype; log 2 [fold-change] > 1, P < 0.05, 2,410 genes) identified via RNA-Seq and genes upregulated in shHDAC5-treated PANC-1 cells ( n = 3 per condition; log 2 [fold-change] > 1, P < 0.05, 2,683 genes), revealing a shared subset of 378 genes. ( B ) Bar graph showing the top 10 enriched transcription factors from transcription factor analysis of 378 genes in A using Enrichr. ATF2, activating transcription factor 2. ( C ) Dual-luciferase reporter assays were performed to assess the transcriptional activity of c-Myc in HDAC5-depleted PANC-1 and AsPC-1 cells. Data are presented as mean ± SD ( n = 5). Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test. *** P < 0.001. ( D ) Heatmap of MYC ChIP-Seq signal intensity (±3 kb around MYC binding sites) in control vs. HDAC5-knockdown PANC-1 cells. ( E ) The average ChIP signal of MYC centered at transcription start site (±3 kb) in indicated groups. ( F ) Venn diagram depicting the overlap between genes with enhanced MYC promoter occupancy after shHDAC5 knockdown, as identified by ChIP-Seq, and genes upregulated in PANC-1 cells following shHDAC5 knockdown, as determined by RNA-Seq. P = 6 × 10 –71 . ( G ) KEGG pathway enrichment analysis of 821 intersecting genes in F . ( H and I ) Western blot analysis of canonical MAPK pathway protein expression under indicated conditions.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: RNA Sequencing, Luciferase, Activity Assay, ChIP-sequencing, Binding Assay, Control, Knockdown, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A and B ) Western blot ( A ) and RT-qPCR ( B ) analyses of c-Myc expression in PANC-1 and AsPC-1 cells infected with indicated shRNAs for 48 hours ( n = 3). ( C ) Western blot analysis of c-Myc protein levels in PANC-1 and AsPC-1 cells transfected with indicated plasmids and treated with DMSO or MG132 (10 μM, 8 hours). EV, empty vector. ( D ) RT-qPCR analysis of c-Myc mRNA in PANC-1 and AsPC-1 cells transfected with indicated plasmids for 48 hours ( n = 3). ( E ) Immunofluorescence analysis of c-Myc in PANC-1 cells transfected with indicated plasmids for 48 hours. Representative images and fluorescence intensity quantification are shown ( n = 5). ( F ) Western blot analysis and quantification of c-Myc protein stability in control or HDAC5-knockdown PANC-1 and AsPC-1 cells treated with cycloheximide (CHX, 50 μg/mL) for indicated times ( n = 3). ( G ) Colocalization analysis of the merged images in E , showing pixel intensity profiles along the white line from left to right in each panel. Colors correspond to the merged images: green for c-Myc and blue for DAPI. All data are presented as the mean ± SD. Statistical significance was determined by 1-way ANOVA followed by Dunnett’s multiple comparisons test ( B , D , and E ) or 2-way ANOVA followed by Tukey multiple comparisons test ( F ). ** P < 0.01, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Infection, Transfection, Plasmid Preparation, Immunofluorescence, Fluorescence, Control, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Co-IP assay showing interaction between c-Myc and HDAC5 in PANC-1 and AsPC-1 cells. ( B ) Schematic diagrams of the truncations of GST-HDAC5. ( C ) Western blot analysis of full-length c-Myc protein in PANC-1 whole-cell lysate pulled down by GST or GST-HDAC5 recombinant proteins. Arrows indicate expected bands. ( D ) Co-IP detection of acetylated lysine on c-Myc in HDAC5-deficient PANC-1 and AsPC-1 cells. ( E ) Co-IP detection of acetylated lysine on c-Myc in PANC-1 and AsPC-1 cells overexpressing plasmids as indicated. ( F and G ) Co-IP analysis of c-Myc ubiquitination levels in HDAC5-deficient ( F ) or HDAC5-overexpressing ( G ) PANC-1 cells. ( H ) A hypothetical model depicting that HDAC5 deacetylates c-Myc, promoting its ubiquitination and degradation.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Recombinant, Ubiquitin Proteomics

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Illustration of c-Myc acetylation at K148 identified by mass spectrometry. ( B ) Mass spectrometry quantification of c-Myc-K148ac intensity in siControl vs. siHDAC5 groups. si, siRNA. ( C ) Different peptides dissolved in double-distilled H 2 O were applied to the nitrocellulose membrane, followed by immunoblotting with anti–c-Myc-K148ac antibody. ( D ) Co-IP detection of c-Myc-K148ac in PANC-1 cells expressing c-Myc (WT/K148R/K148Q) and treated with shNC or shHDAC5. ( E – G ) PANC-1 and AsPC-1 cells were transfected with equal amounts of c-Myc (WT/K148R/K148Q) plasmids for 48 hours. Afterward, cells were harvested for RT-qPCR analysis ( E ) and Western blot analysis ( F ), followed by protein quantification analysis ( G ) ( n = 3). ( H ) Western blot analysis was conducted to evaluate c-Myc protein levels and normalized protein intensity in PANC-1 cells transfected with equal amounts of c-Myc (WT/K148R/K148Q) plasmids after treatment with 50 μg/mL CHX for the indicated durations ( n = 3). ( I ) Co-IP detection of c-Myc ubiquitination in PANC-1 cells expressing c-Myc (WT/K148R/K148Q) and treated with shNC or shHDAC5. ( J ) Illustration of c-Myc ubiquitination at K148 identified by mass spectrometry. ( K ) A bar graph showing the intensities of c-Myc ubiquitination at K148 identified by mass spectrometry in shNC and shHDAC5 groups. ( L ) Luciferase reporter activities of c-Myc were assessed in PANC-1 cells infected with shHDAC5 and knockin c-Myc (WT/K148R/K148Q) ( n = 5). ( M ) A hypothetical model illustrating how HDAC5 regulates c-Myc degradation through the competition between K148 acetylation and ubiquitination. All data are presented as the mean ± SD. Statistical significance was determined by 1-way ANOVA ( E and G ) or 2-way ANOVA ( H and L ), followed by Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Mass Spectrometry, Membrane, Western Blot, Co-Immunoprecipitation Assay, Expressing, Transfection, Quantitative RT-PCR, Ubiquitin Proteomics, Luciferase, Infection, Knock-In

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Mass spectrometry identified NEDD4 peptide fragments coprecipitated with c-Myc antibody, showing reduced signals upon HDAC5 knockdown. ( B and C ) Western blot analysis of PANC-1 cells infected with indicated siRNAs for 48 hours. ( D ) Western blot analysis and quantification of c-Myc protein stability in control or NEDD4-knockdown PANC-1 cells after CHX (50 μg/mL) treatment. All data are presented as the mean ± SD ( n = 3). Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test. * P < 0.05. ( E ) Endogenous co-IP analysis of the NEDD4/c-Myc interaction in PANC-1 cells transfected with indicated siRNAs. ( F and G ) Co-IP detection of c-Myc ubiquitination ( F ) and c-Myc-K148ac ( G ) in PANC-1 cells expressing c-Myc-WT and treated with siHDAC5, siNEDD4, or both. ( H ) Co-IP detection of c-Myc ubiquitination in PANC-1 cells expressing c-Myc (WT/K148R/K148Q) and treated with siControl or siNEDD4. ( I ) In vitro ubiquitination assay evaluating NEDD4 regulation of c-Myc (WT/K148R/K148Q) ubiquitination at K148. ( J ) A hypothetical model illustrating how HDAC5 and NEDD4 competitively modify the acetylation and ubiquitination of c-Myc at the K148 site, collaboratively promoting c-Myc protein degradation.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Mass Spectrometry, Knockdown, Western Blot, Infection, Control, Co-Immunoprecipitation Assay, Transfection, Ubiquitin Proteomics, Expressing, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: HDAC5 deficiency induces intrinsic resistance to KRAS inhibition by disrupting c-Myc acetylation-ubiquitination homeostasis

doi: 10.1172/JCI195814

Figure Lengend Snippet: ( A ) Representative macroscopic images of pancreatic tumors from KPC Hdac5-WT and KPC Hdac5-KO mice after sacrifice. ( B ) Tumor weight analysis in KPC mice treated with vehicle, MRTX1133 (30 mg/kg, i.p. bid), MYCi975 (50 mg/kg, i.p. bid), or their combination ( n = 5). ( C ) Kaplan-Meier survival curves with log-rank test ( n = 5). *** P < 0.001. ( D and E ) Representative IHC images of tumors from treated mice and quantified IHC scores ( E ). Scale bars = 100 μm. n = 5 biologically independent repeats and 3 independent quantifications. ( F and G ) Representative bioluminescence images and quantification ( n = 5). ( H ) Macroscopic images of PDX tumors after 30 days of treatment. ( I and J ) Tumor growth curves ( I ) and final tumor weights ( J ) in PDX models treated with Vehicle + LV-Control, MRTX1133 (30 mg/kg, i.p. bid) + LV-Control, Vehicle + LV-sgMYC (50 μL lentivirus, s.c., weekly), or MRTX1133 + LV-sgMYC. All data are presented as the mean ± SD. Statistical significance was determined by 2-way ANOVA followed by Tukey’s multiple comparisons test ( B , E , G , I , and J ). *** P < 0.001.

Article Snippet: KP [ Kras tm1(LSL-G12D) Trp53 tm1(LSL-R172H) , C001320] mice, Hdac5 -KO (S-KO-02424) mice, and Tg( Pdx1-Cre ) (C001408) mice were all purchased from Cyagen and interbred to generate KPC Hdac5-KO mice.

Techniques: Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A, VS-6063 reduced levels of phosphorylated HDAC4 and HDAC5. n=4 biological replicates in each group. B, Silencing FAK also reduced the phosphorylation of HDAC4 and HDAC5 in HASMCs grown in common medium or osteogenic medium. n=4 biological replicates in each group. C, FAK inhibition using VS6063 decreased cytosolic localization and increased nuclear localization of HDAC4 and HDAC5 in osteogenic media as shown by immunofluorescence images (60x). 30 cells in each group were used for analysis.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Phospho-proteomics, Immunofluorescence

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: Leptomycin B (LEP) inhibited nuclear export of HDAC4 and HDAC5 in A , normal media and B , in osteogenic media as shown by immunofluorescence. 12–46 cells in each group were used for analysis. C, Leptomycin B reduced phosphorylated HDAC4 and HDAC5 and inhibited the expression of RUNX2 and ALPL in osteogenic media. n=4 biological replicates in each group.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Immunofluorescence, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A , Increased expression of HDAC4/5 was achieved by adenovirus transduction (Ad.HDAC4 or Ad.HDAC5). B, Ad.HDAC4 or Ad.HDAC5 resulted in enhanced calcification that was inhibited with the treatment of leptomycin B (5nM) n=3 biological replicates in each group (with 2 representative replicates shown). Quantitative calcium assay in cells treated with C, Ad.HDAC4 and D , Ad.HDAC5 in the presence or absence of leptomycin demonstrated reduced calcification with leptomycin treatment. n=3 biological replicates in each group. E and F, Cytosolic and nuclear localization of HDAC4 and HDAC5 in AdHDAC4 and AdHDAC5 treated cells in the presence and absence of leptomycin (a nuclear export inhibitor, 10 nM for 3 hours) in osteogenic media. 12–13 cells in each group were used for analysis.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Over Expression, Expressing, Transduction, Calcium Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A, Treatment of HASMCs with siHDAC4 resulted in >70% and >65% knockdown of HDAC4 mRNA level in common or osteogenic medium, respectively. n=6 biological replicates in each group. B, Protein levels of RUNX2 and ALPL were increased with osteogenic medium. However, siHDAC4 decreased the levels of RUNX2 and ALPL induced by osteogenic medium. n=4 biological replicates in each group. C, Treatment of HASMCs with siHDAC5 resulted in >55% and >50% knockdown of HDAC5 mRNA level in normal or osteogenic medium, respectively, and decreased HDAC5 protein levels in siHDAC5-treated cells. n=6 biological replicates in each mRNA group. D, siHDAC5 decreased the protein levels of RUNX2 and ALPL induced by osteogenic medium. n=4 biological replicates in each group. E, Treatment with siHDAC4, siHDAC5, or the combination inhibited calcification of HASMCs grown in osteogenic medium for 14 days, as evidenced by Alizarin Red staining. n=3 biological replicates in each group (with 2 representative replicates shown). F, Treatment of HASMCs with LMK-235 (a pharmacologic inhibitor of HDAC4 and HDAC5) inhibited calcification induced by osteogenic medium in a dose-dependent manner. n=2 biological replicates in each group G, LMK-235 reduced the migration of VSMCs induced by osteogenic medium. The experiments in figure 1H and 7G were performed at the same time. n=6 biological replicates in each group.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Knockdown, Staining, Migration

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: A and B, Increased HDAC4 or HDAC5 expression by adenovirus resulted in augmented calcification of HASMCs that was inhibited by treatment with the FAK inhibitor VS-6063 (1μM). n=3 biological replicates in each group (with 2 representative replicates shown). C and D, Reduction of FAK expression with siFAK significantly attenuated the calcification of HASMCs induced by Ad.HDAC4 or Ad.HDAC5 in osteogenic medium or induced by osteogenic medium alone. n=3 biological replicates in each group (with 2 representative replicates shown). E and F, Osteogenic medium induced calcification of mouse aortas and human carotid arteries after culturing for 21 days. However, the calcification induced by osteogenic medium was inhibited by treatment with VS-6063 (2μM or 4μM). n=4 biological replicates in each group.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Inhibition, Over Expression, Cell Culture, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Focal adhesion kinase promotes calcification of vascular smooth muscle cells via regulation of histone deacetylase 4 and 5

doi: 10.1161/ATVBAHA.123.319010

Figure Lengend Snippet: Localization of PTK2 , HDAC5 , and HDAC4 gene expression in modulated SMC subtypes using an integrated human atherosclerosis reference. Uniform Manifold Approximation and Projection (UMAP) embeddings from an integrated human atherosclerosis single-cell RNA-seq reference dataset (see Methods ), highlighting ( A ) PTK2 , ( B ) HDAC5 , and ( C ) HDAC4 normalized gene expression. Individual sequencing libraries across four studies were harmonized after QC and batch correction with reciprocal PCA (rPCA). A broad SMC cluster was annotated using transfer learning with cell labels from the Tabula Sapiens vasculature subset. SMC subtypes were further annotated by extracting gene modules from a scRNA meta-analysis of murine SMCs (contractile SMC, transitional SMC, fibromyocyte, and fibrochondrocyte) and calculating their enrichment in cells within the main SMC cluster. PTK2 and HDAC5 expression were enriched in transitional SMCs and fibromyocytes.

Article Snippet: The primary antibodies in our study were FAK (1:1000, #3285, Cell Signaling Technology), p-FAK (Y397) (1:1000, #8556, Cell Signaling Technology), HDAC4 (1:1000, #17449, Proteintech), HDAC5 (1:1000, #16166, Proteintech), phospho-HDAC4/HDAC5 (1:1000, #3443, Cell Signaling Technology), RUNX2 (1:1000, #12556, Cell Signaling Technology), ALPL (1:1000, #11187, Proteintech), GAPDH (1:1000, #2118S, Cell Signaling Technology).

Techniques: Gene Expression, RNA Sequencing, Sequencing, Expressing