Journal: EBioMedicine

Article Title: Ketogenesis acts as an endogenous protective programme to restrain inflammatory macrophage activation during acute pancreatitis

doi: 10.1016/j.ebiom.2022.103959

Figure Lengend Snippet: βOHB limits proinflammatory macrophage activation via class I HDACs, (a, b) Gene set enrichment analysis (GSEA) of gene expression in murine BMDMs are depicted. (a) Enrichment of TSA-upregulated signature genes (GSE22049) in βOHB-treated BMDMs. (b) Enrichment of HDAC3 depletion related signature genes (GES33162) in βOHB-treated BMDMs. (c, d) BMDMs treated with LPS (100 ng/ml) together with βOHB (10 mM), TSA (1 μΜ), or MS-275 (20 μM) for 4 h. (c) Flow cytometry analysis of intracellular TNFα levels. (d) Quantitative PCR analysis of Nos2, Il6, Il12b , and Tnfa mRNA expression. (e) ChIP-qPCR analysis of H3Ac and H4Ac occupancy in Edn1, Il12a , and Nos2 loci in the indicated group, with IgG as the control. (f) Ingenuity Pathway Analysis (IPA) of genes in cluster A. (g) BMDMs were pretreated with βOHB (10 mM) or TSA (1 μΜ) 1 h prior to LPS (100 ng/ml) stimulation for 15 min. Acylated p65 levels were determined by immunoprecipitation, while p-p65 and total p65 were determined by immunoblotting. (h) Mice were i.p. injected with TSA (0.1 mg/kg), MS-275 (20 mg/kg) or DMSO as a control 1 h prior to the CER-AP (Cer*7) induction and were harvested at 24 h thereafter (n = 5 / group). Frequency of TNFα + macrophagess (F4/80 + CD11b + ) in pancreatic leukocytes from the indicated groups. Data are presented as the mean ± SD in bar graphs (c–e) or the mean ± SEM in dot plots (h). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: For treatments, the Balb/c mice were (1) fed with 1,3-butanediol (5.72% (w/v); Sigma-Aldrich) or normal water (control) one week before disease induction; received a single intraperitoneal injection of βOHB (3 mmol/kg; Sigma-Aldrich) or normal saline at the indicated time; and (3) the CPT1α inhibitor etomoxir (20 mg/kg, Sigma-Aldrich), histone deacetylase (HDAC) pan inhibitor TSA (0.1 mg/kg; Targetmol), HDAC1/3 inhibitor MS-275 (20 mg/kg; Targetmol), or dimethyl sulfoxide (DMSO) as a control 1 h before disease induction.

Techniques: Activation Assay, Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Injection

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: APPL1 is a part of the β-catenin-Reptin-HDAC1/2 complex. A , HEK293 cell lysates were subjected to a coimmunoprecipitation assay ( IP ) using anti-Reptin or control IgG, and the resultant precipitates were subjected to immunoblotting ( IB ) with the indicated antibodies. TCL , total cell lysate. B , a 2D BN/SDS-PAGE analysis of HEK293 cell extracts. Multiprotein complexes were resolved on 6–13% acrylamide gradient gel, followed by Tris-glycine-SDS-PAGE (8% gel). Immunoblotting was performed with specific antibodies recognizing the indicated proteins.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Co-Immunoprecipitation Assay, Western Blot, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: Overexpression of APPL proteins reduces the amounts of HDACs bound to Reptin. A , HEK293 cells were transiently transfected with APPL1-Myc and APPL2-Myc. Forty-eight hours post-transfection, cell extracts were prepared, and a coimmunoprecipitation assay ( IP ) was performed with either anti-Reptin or control IgG. The resultant precipitates were analyzed by immunoblotting ( IB ) against the indicated antibodies ( A ) or were subjected to fluorometric assays for measuring the respective HDAC activities ( C ). Each HDAC activity assay was performed in duplicate, and relative fluorescence unit ( RFU ) values shown are the averages ± S.D. from which the RFU values of assay buffer were subtracted. B , HEK293 cells were cotransfected with plasmids encoding YFP-tagged APPL1 or empty vector. Cell extracts were prepared and immunoprecipitated using anti-HDAC2. Bound proteins were separated and visualized by immunoblotting using anti-HDAC2, anti-HDAC1, anti-Reptin, and anti-APPL1 antibodies.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, HDAC Activity Assay, Fluorescence, Plasmid Preparation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription *

doi: 10.1074/jbc.M109.007237

Figure Lengend Snippet: Recruitment of β-catenin, Reptin, and HDAC1 to the promoters of Wnt target genes is affected upon overexpression of APPL proteins. A , overexpression of APPL proteins reduces the amount of Reptin bound to β-catenin. HEK293 cells were transiently transfected with APPL1 or APPL2. Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h. Subsequently, cell extracts were prepared, and a coimmunoprecipitation assay ( IP ) was performed with anti-β-catenin or control IgG. The resulting precipitates were analyzed by immunoblotting ( IB ) with the indicated antibodies. B , APPL proteins increase the recruitment of β-catenin and reduce the amounts of Reptin and HDAC1 at the Wnt target gene promoters. HEK293 cells were transfected with either empty pcDNA 3.1 ( lane 1 ) or plasmids expressing APPL1 ( lane 2 ) and APPL2 ( lane 3 ). Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h and then subjected to chromatin immunoprecipitation using anti-β-catenin, anti-HDAC1, and anti-Reptin antibodies. PCR was performed from these immunoprecipitates by using the primer pair covering the β-catenin binding sites at the promoters of cyclin D1 and Axin2. PCR products were resolved by agarose gel and stained with ethidium bromide. Lane c , PCR mixture without template.

Article Snippet: The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-β-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology).

Techniques: Over Expression, Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis, Staining

Journal: PLoS Pathogens

Article Title: Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

doi: 10.1371/journal.ppat.1000965

Figure Lengend Snippet: (A) The interaction between pUL38 and HDAC1 requires additional factors present during viral infection. Immunoprecipitation (IP) from whole cell lysates isolated from either uninfected control cells (HF) or pUL38-expressing cells (HF-UL38) using antibodies against HDAC1 (lanes 1, 4) or TSC2 (lane 6). Immunoprecipitation of HDAC1 (lane 5) was also completed upon infection of HF-UL38 cells with a pUL38-deficient virus, BAD sub UL38 ( sub UL38) at 24 hpi. Binding to pUL38 was detected by Western blot (WB) analysis with an anti-pUL38 antibody. Beads without antibody and normal IgG were used as specificity controls (lane 2, 3). (B) pUL38 binding to HDAC1 requires pUL29/28. Cells were infected at 3 pfu/cell using wild-type (BAD wt ), BAD sub UL29 ( sub UL29) or BAD sub UL28 ( sub UL28) mutant viruses and whole cell lysates collected at 24 hpi. Interactions were detected by immunoprecipitation of HDAC1 and Western blot to pUL38. Lysate controls demonstrate expression of pUL38 in all samples. (C) pUL29 binds HDAC1 in the absence of infection. Expression vectors pCGN or pCGN-pUL29HA (pUL29HA) were transfected into U-2 OS cells and whole cell lysates were collected 48 h later. Binding was demonstrated by immunoprecipitation of HDAC1 and Western blot to HA. Lysate controls show expression of pUL29HA, HDAC1 and tubulin, and an asterisks indicates heavy chain of IgG.

Article Snippet: Antibodies used in these studies were: mouse anti-HDAC1 IgG1 (Millipore), rabbit anti-HDAC1 polyclonal (Millipore), rabbit anti-MTA2 polyclonal (Santa Cruz), mouse anti-FLAG M2 IgG1 (Sigma), mouse anti-Myc IgG1(Millipore), rabbit anti-mSin3A polyclonal (Santa Cruz), rabbit anti-TSC2 polyclonal (Santa Cruz), mouse anti-HA IgG1 (Sigma), mouse anti-tubulin IgG1 (Sigma), rabbit anti-protein A (Sigma), mouse anti-IE1 (clone 1B12) mouse anti-pUL38 (clone 8D6) and mouse anti-pUS24 .

Techniques: Infection, Immunoprecipitation, Isolation, Control, Expressing, Virus, Binding Assay, Western Blot, Mutagenesis, Transfection

Journal: Cells

Article Title: Targeting Class I Histone Deacetylases in Human Uterine Leiomyosarcoma

doi: 10.3390/cells11233801

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: HDAC1 , Abcam , Ab109411 , Rabbit , IHC , 1:50 , .

Techniques:

Journal: Cells

Article Title: Targeting Class I Histone Deacetylases in Human Uterine Leiomyosarcoma

doi: 10.3390/cells11233801

Figure Lengend Snippet: IHC staining of HDAC1, 2, and 3 in human uLMS tissues and adjacent myometrium. IHC staining for HDAC1, 2, and 3 is presented with three representative cases. The right column showed the density map of HDAC1, 2, and 3 for the same representative case. Blue color: negative; Yellow color: low expression; brown color: moderate expression; red color: strong expression. Scale bars in black color: 100 µm; Scale bars in red color: 1mm.

Article Snippet: HDAC1 , Abcam , Ab109411 , Rabbit , IHC , 1:50 , .

Techniques: Immunohistochemistry, Expressing

Journal: Cells

Article Title: Targeting Class I Histone Deacetylases in Human Uterine Leiomyosarcoma

doi: 10.3390/cells11233801

Figure Lengend Snippet: Percentage of HDACs positive cells and H-score of HDACs expression in uLMS vs. myometrium. ( A ) Percentage of HDAC1, HDAC2, and HDAC3 positive cells in uLMS and myometrium tissues; ( B ) H-score of HDAC1, HDAC2, and HDAC3 in uLMS and myometrium tissues. * p < 0.05. *** p < 0.001. ns: no significant difference.

Article Snippet: HDAC1 , Abcam , Ab109411 , Rabbit , IHC , 1:50 , .

Techniques: Expressing

Journal: Cells

Article Title: Targeting Class I Histone Deacetylases in Human Uterine Leiomyosarcoma

doi: 10.3390/cells11233801

Figure Lengend Snippet: The expression of Class I HDACs in UTSM, HuLM, MES-SA, and SK-UT-1 cell lines . The protein levels of Class I HDACs (HDAC1 [( A ), 2 ( B ), 3 ( C ), and 8 ( D )] were measured by Western blot. β-actin was used as an endogenous control. Quantitative analysis of relative levels of HDAC1, 2, 3, and 8 ( A – D ) was performed using Image J (1.53t version) (NIH, Bethesda, MD, USA). *** p < 0.001.

Article Snippet: HDAC1 , Abcam , Ab109411 , Rabbit , IHC , 1:50 , .

Techniques: Expressing, Western Blot

Journal: Cell reports

Article Title: PRMT1 promotes epigenetic reprogramming associated with acquired chemoresistance in pancreatic cancer

doi: 10.1016/j.celrep.2024.114176

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-HDAC1 , Proteintech , Cat#10197–1-AP; RRID:AB_2920338.

Techniques: Recombinant, cDNA Synthesis, SYBR Green Assay, Bicinchoninic Acid Protein Assay, DNA Extraction, Sequencing, Gene Expression, Plasmid Preparation, Software