hdac7 antibody  (Cell Signaling Technology Inc)

Journal: American journal of physiology. Cell physiology

Article Title: Activation of the Piezo1 channel stimulates protein kinase D and migration in human aortic endothelial cells

doi: 10.1152/ajpcell.00457.2025

Figure Lengend Snippet: A, Confluent ECs were incubated with PKD inhibitor CRT0066101 at 2.5 μM (CRT) for 1h prior to stimulation with 5μM Yoda1 for 10 min. The cultures were lysed with 2X SDS-PAGE sample buffer and analyzed by immunoblotting with antibodies that detect HDAC7 phoshorylated at Ser 486 , PKD1 phosphorylated at Ser 910 , HDAC7 and GAPDH (as a loading control). B , Bars display the quantification of HDAC7 phosphorylation. Mean ± SEM; n=3 independent experiments, **p<0.001 by Student’s t-test. C , Confluent ECs were treated without or with 2.5 μM CRT0066101 (CRT) for 1 h and then stimulated with Yoda1 for 60 min, as indicated. ECs were fixed and stained with an antibody that detects HDAC7 and images obtained by confocal microscopy. Scale bar = 15μm. D, Bars represent the percentage of ECs treated with Yoda1 (Y1), CRT0066101 (CRT) or their combination (Y1+CRT) with nuclear HDAC7 obtained from 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. E, Ratio of nuclear/cytoplasmic HDAC7 fluorescence in 5–6 fields with a total of ~30 individual cells in each field, mean ± SEM; n= 5 fields, **p<0.001 control vs Y1 and ## p<0.001 Y1 vs Y1 +CRT. The treatments were as described in panel D. F, Confluent ECs were incubated with 10 μg/ml mitomycin C for 2 h. A scratch wound was then created with a sterile 200-μl pipette tip and after washing, wounded cells were stimulated with Yoda1 or Yoda2 at 5μM for 18 h. The cultures were fixed with 4% paraformaldehyde and stained with Giemsa stain. Representative microscopy fields are shown. Images were captured at original magnification 10X. G, Bars represent relative migration (average of 5 fields per experiment) of ECs stimulated with Yoda1 or Yoda2 in the absence or presence of CRT0066101 (1 μM and 2.5 μM). Bars represent the means ± SEM n= 4 for Yoda1 and n=3 for Yoda2 independent experiments., *p<0.02, **p,0.01 by Student’s t-test.

Article Snippet: Piezo1 antibody (Proteintech Cat# 15939–1-AP, RRID:AB_2231460; final dilution 1:1000); HDAC7 antibody ( Proteintech Cat# 26207–1-AP, RRID:AB_288042626207; final dilution 1:1000) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217; final dilution 1:1000) were from Thermo Fisher Scientific (Waltham, MA).

Techniques: Activation Assay, Phospho-proteomics, Migration, Incubation, SDS Page, Western Blot, Control, Staining, Confocal Microscopy, Fluorescence, Sterility, Transferring, Giemsa Stain, Microscopy

Journal: Hepatology Communications

Article Title: ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

doi: 10.1097/HC9.0000000000000738

Figure Lengend Snippet: ARID1A deficiency renders upregulation of HDAC7 expression in HCC cells. (A) GO enrichment analysis of upregulated differentially expressed genes between shARID1A or control group. (B) Heatmap depicting relative expression of the top 25 differentially expressed genes in the chromatin modification pathway between the shARID1A and control group. (C) Fold changes in mRNA expression of HDAC family genes from RNA-seq between the shARID1A versus control group. (D and E) Relative mRNA expression of ARID1A and HDACs in Huh7 or HepG2 cells± ARID1A knockdown. (F and G) Western blot of HDAC7 in HepG2 or Huh7 cells± ARID1A knockdown. (H) Relative mRNA expression of ARID1A and HDACs in SNU449 cells± ARID1A overexpression. (I) Western blot of HDAC7 in SNU449 cells± ARID1A overexpression. (J and K) Western blot of pan-acetylation level in Huh7 or HepG2 cells± ARID1A knockdown. (L) Western blot of pan-acetylation level in SNU449 cells± ARID1A overexpression. qRT-PCR data (mean±SD; n=3) were analyzed by unpaired 2-tail t test. * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; GO, Gene Ontology; HDAC7, histone deacetylase 7; WT, wild type.

Article Snippet: Antibodies against β-actin (#81115-1-RR), HDAC7 (for IP, #26207-1-AP), and ENO1 (#11204-1-AP) were purchased from Proteintech.

Techniques: Expressing, Control, Modification, RNA Sequencing, Knockdown, Western Blot, Over Expression, Quantitative RT-PCR, Histone Deacetylase Assay

Journal: Hepatology Communications

Article Title: ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

doi: 10.1097/HC9.0000000000000738

Figure Lengend Snippet: HDAC7 is upregulated in HCC, and inhibition of HDAC7 restrains the proliferation of ARID1A-deficient cells. (A) Box plots showing the expression of HDAC7 mRNA in 369 HCC tumor samples and 160 corresponding normal samples from the TCGA database using the GEPIA website. (B) Kaplan-Meier survival analysis of HDAC7 expression in 364 HCC cases from the TCGA database using the GEPIA website. (C) IHC staining of ARID1A and HDAC7 protein in HCC tissues and the matched noncancerous liver tissues. Shown are representative images. Scale bar, 100 μm. (D) Correlation of H-scores for ARID1A and HDAC7 proteins in HCC samples and the paired noncancerous liver tissues, n=15. The correlation score was analyzed using the Pearson correlation test. (E and F) Proliferation was measured by EdU incorporation assay of Huh7 or HepG2 cells± ARID1A knockdown treated with or without 2 μM TMP269 for 24 hours. Nuclei were counterstained with Hoechst. Scale bar, 100 μm. G and H, Proliferation was measured by CCK8 assay of Huh7 or HepG2 cells± ARID1A knockdown treated with or without 2 μM TMP269 for 24 hours. Data (mean±SD; n=3) were analyzed by repeated-measures ANOVA. I and J, Proliferation was measured by colony formation assay of Huh7 or HepG2 cells± ARID1A knockdown treated with or without 2 μM TMP269 for 24 hours. * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; HDAC7, histone deacetylase 7.

Article Snippet: Antibodies against β-actin (#81115-1-RR), HDAC7 (for IP, #26207-1-AP), and ENO1 (#11204-1-AP) were purchased from Proteintech.

Techniques: Inhibition, Expressing, Immunohistochemistry, Knockdown, CCK-8 Assay, Colony Assay, Histone Deacetylase Assay

Journal: Hepatology Communications

Article Title: ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

doi: 10.1097/HC9.0000000000000738

Figure Lengend Snippet: ARID1A deficiency promotes PU.1-mediated transcriptional regulation of HDAC7 in HCC cells. (A) Occupancy of ARID1A with the HDAC7 promoter was determined by ChIP-PCR in Huh7 cells. (B) Chromatin accessible sites in HDAC7 promoter were determined by ATAC-seq in Huh7 cells± ARID1A knockdown. (C) Transcription factors with potential binding sites with the HDAC7 promoter were ranked by credibility score from UCSC and the JASPAR data sets. (D–F) Western blot of HDAC7 in Huh7 cells± STAT2, SPI1 , or IRF1 knockdown. (G) Occupancy of PU.1 with the HDAC7 promoter was determined by ChIP-PCR in Huh7 cells. (H) Relative occupancy of PU.1 with the HDAC7 promoter was determined by ChIP-qPCR in Huh7 cells. (I) Promoter activity of HDAC7 in HEK293T cells overexpressing PU.1 was examined by luciferase reporter assay. (J and K) Western blot of HDAC7 in Huh7 or HepG2 cells± ARID1A and/or SPI1 knockdown. (L and M) co-IP analysis of the interaction between ARID1A and PU.1 in Huh7 or HepG2 cells. (N and O) Localization of ARID1A and PU.1 protein in Huh7 or HepG2 cells was examined by immunofluorescence assay. Scale bar, 10 μm. (P) Relative occupancy of PU.1 with the HDAC7 promoter was determined by ChIP-qPCR in Huh7 cells± ARID1A knockdown. ChIP-qPCR and luciferase reporter data (mean±SD; n=3) were analyzed by unpaired 2-tail t test. * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; HDAC7, histone deacetylase 7.

Article Snippet: Antibodies against β-actin (#81115-1-RR), HDAC7 (for IP, #26207-1-AP), and ENO1 (#11204-1-AP) were purchased from Proteintech.

Techniques: Knockdown, Binding Assay, Western Blot, ChIP-qPCR, Activity Assay, Luciferase, Reporter Assay, Co-Immunoprecipitation Assay, Immunofluorescence, Histone Deacetylase Assay

Journal: Hepatology Communications

Article Title: ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

doi: 10.1097/HC9.0000000000000738

Figure Lengend Snippet: ARID1A deficiency promotes HCC cell proliferation through the HDAC7-ENO1 axis. (A) Coomassie brilliant blue-stained gel showed differential bands between control and HDAC7-overexpressing samples in Huh7 cells. (B) Screening of ENO1 as a potential substrate of HDAC7. (C and D) co-IP analysis of the interaction between HDAC7 and ENO1 in Huh7 or HepG2 cells. (E and F) Western blot of Ac-ENO1 in Huh7 or HepG2 cells± HDAC7 knockdown. (G and H) Western blot of Ac-ENO1, cyclin D1, and p21 in Huh7 or HepG2 cells± HDAC7 knockdown treated with or without ENO1-K89R mutant. (I and J) Proliferation was measured by EdU incorporation assay of Huh7 or HepG2 cells± ARID1A knockdown treated with or without ENO1-K89R mutant. Nuclei were counterstained by Hoechst. Scale bar, 100 μm. (K and L) Western blot of Ac-ENO1, ENO1, cyclin D1, and p21 in Huh7 or HepG2 cells± ARID1A knockdown treated with 2 μM TMP269 for 24 hours, ENO1-K89Q mutant or ENO1-K89R mutant. (M and N) Proliferation was measured by EdU incorporation assay of Huh7 or HepG2 cells± ARID1A knockdown treated with 2 μM TMP269 for 24 hours, ENO1-K89Q mutant, or ENO1-K89R mutant. Nuclei were counterstained with Hoechst. Scale bar, 100 μm. Abbreviations: ARID1A, AT-rich interaction domain 1A; EdU, 5‑ethynyl‑2′‑deoxyuridine; ENO1, enolase 1; HDAC7, histone deacetylase 7.

Article Snippet: Antibodies against β-actin (#81115-1-RR), HDAC7 (for IP, #26207-1-AP), and ENO1 (#11204-1-AP) were purchased from Proteintech.

Techniques: Staining, Control, Co-Immunoprecipitation Assay, Western Blot, Knockdown, Mutagenesis, Histone Deacetylase Assay

Journal: Hepatology Communications

Article Title: ARID1A deficiency promotes malignant proliferation of hepatocellular carcinoma by activating HDAC7/ENO1 signaling pathway

doi: 10.1097/HC9.0000000000000738

Figure Lengend Snippet: Targeted inhibition of HDAC7 inhibits the progression of ARID1A-deficient HCC in vivo. (A) Image of subcutaneous tumors derived from Huh7 cells± ARID1A knockdown treated with PBS, 3 mg/kg/d TMP269, and 20 mg/kg/d SAHA at the end of scheduled treatment. (B) Growth curves of subcutaneous tumors derived from Huh7 cells± ARID1A knockdown treated with PBS, 3 mg/kg/d TMP269, and 20 mg/kg/d SAHA. Data (mean±SD; n=3) were analyzed by repeated-measures ANOVA. (C) IHC staining of ARID1A and HDAC7 protein of subcutaneous tumors derived from Huh7 cells± ARID1A knockdown treated with PBS, 3 mg/kg/d TMP269, and 20 mg/kg/d SAHA. Scale bar, 100 μm. (D and E) Quantification of IHC staining of AIRD1A and HDAC7 in C. Data (mean±SD; three animals in each group) were analyzed by unpaired 2-tail t test. F, Schematic representation of the molecular mechanism of ARID1A deficiency promoting progression of HCC through upregulating HDAC7. * p <0.05; ** p <0.01; *** p <0.001; **** p <0.0001. Abbreviations: ARID1A, AT-rich interaction domain 1A; ENO1, enolase 1; HDAC7, histone deacetylase 7.

Article Snippet: Antibodies against β-actin (#81115-1-RR), HDAC7 (for IP, #26207-1-AP), and ENO1 (#11204-1-AP) were purchased from Proteintech.

Techniques: Inhibition, In Vivo, Derivative Assay, Knockdown, Immunohistochemistry, Histone Deacetylase Assay

Journal: The Journal of Physiology

Article Title: Muscle regeneration is improved by hot water immersion but unchanged by cold following a simulated musculoskeletal injury in humans

doi: 10.1113/jp287777

Figure Lengend Snippet: Figure 8. Representative blots and quantification of phosphorylated NF-κB p65 (A, B) and IL-10 (C, D) protein expression at PRE, D5 and D11 in CWI, TWI and HWI groups Data are presented as medians along with individual data. #Significantly different from PRE (P < 0.05); ##significantly different from PRE (P < 0.01); †significantly different from D5 (P < 0.05); ∗significantly different between groups (P < 0.05).

Article Snippet: The membranes were incubated overnight at 4°C with the following primary antibodies: rabbit polyclonal phosphorylated nuclear factor-κB (NF-κB) p65 (1:1000, cat. no. 3033, Cell Signaling Technology, Danvers, MA, USA), monoclonal rat interleukin (IL)-10 (1:500, sc-73309, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal HSP70 (1:750, ADI-SPA-810, Enzo, Farmingdale, NY, USA), mouse monoclonal HSP27 (1:750, ADI-SPA-800, Enzo Life Sciences, Farmingdale, NY, USA), rabbit polyclonal phosphorylated mammalian target of rapamycin (mTOR; 1:1000, cat. no. 2971, Cell Signaling Technology), rabbit polyclonal phosphorylated S6 ribosomal protein (1:1000, cat. no. 5364, Cell Signaling Technology), mousemonoclonal p-p70 S6 kinase (1:1000, cat. no. 9206, Cell Signaling Technology) rabbit polyclonal vascular endothelial growth factor (VEGF; 1:500, sc507, Santa Cruz Biotechnology) and rabbit polyclonal transforming growth factor β (TGF-β 1:1000, cat. no. 3711, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing