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: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 8. ARID1A interacts with HDAC1 through its C-terminal domain. (A) Silver-stained gel shows differential bands between control and ARID1A-overexpressing samples. (B) HEK293T cells were transfected with ARID1A-Flag and HDAC1-HA, and their interaction is examined by co-IP. (C) Interaction between ARID1A and HDAC1 is examined by GST pull-down. (D) The interaction between endogenous ARID1A and HDAC1 is examined by co-IP with HDAC1 antibody in HEK293T and SNU-398 cells. (E) The semi-exogenous interaction between ARID1A and HDAC1 is examined by co-IP with Flag antibody or HDAC1 antibody in HEK293T cells transfected with ARID1A-Flag. (F) Localization of ARID1A and HDAC1 proteins in YY-8103 cells is examined by immunofluorescence assay. Scale bar: 25 mm. (G) The schematic diagram of full-length ARID1A and 5 truncated mutants. (H) Interactions between different ARID1A mutants with HDAC1 in HEK293T cells are examined by co-IP with HA antibody. The arrows indicate exogenous ARID1A with Flag tag. (I) Interactions between different mutants of ARID1A and HDAC1 in PVTT cells are examined by co-IP with Flag antibody. The arrows indicate exogenous Flag-tagged ARID1A. DEL,deletion; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Staining, Control, Transfection, Co-Immunoprecipitation Assay, FLAG-tag

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Targeting USP9X-AMPK Axis in ARID1A-Deficient Hepatocellular Carcinoma.

doi: 10.1016/j.jcmgh.2022.03.009

Figure Lengend Snippet: Figure 9. ARID1A regulates the promoter activity of USP9X via HDAC1. (A) Data from the Catalogue of Somatic Mutations in Cancer shows that 1989* is the most frequent mutation of ARID1A. (B) Interaction between ARID1A-WT or ARID1A-1989* mutation with HDAC1. (C) Influence of ARID1A-WT or ARID1A-1989* mutation on the ubiquitination of PRKAA2. (D) Influence of ARID1A-WT or ARID1A-1989* mutation on the promoter activity of USP9X. The promoter activity of USP9X in (E) HEK293T cells overexpressing ARID1A or HDAC1 (OE) or in (F) ARID1A knockout Huh7 and YY-8103 cells is examined by luciferase reporter assay. (G) Influence of ARID1A-WT or ARID1A-1989* mutation on the expression of USP9X and PRKAA2. CTRL, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. **P<0.01, ***P<0.001, ns, not significant.

Article Snippet: Antibodies against acetyl–histone H3 (Lys9) (9649), acetyl–histone H3 (Lys9) (8173), ULK1 (8054), phospho-ULK1 (Ser317) (12753), phospho-ULK1 (Ser555) (5869), phospho-ULK1 (Ser757) (6888), acetyl-CoA carboxylase (3676), phospho-acetyl-CoA carboxylase (Ser79) (11818), AMPKa (2532), phosphoAMPKa (Thr172) (2535), LC3B (3868), and HDAC1 (34589) were purchased from Cell Signaling Technology (Danvers, MA); antibodies against PRKAA1 (10929), PRKAA2 (18167), USP9X (55054), glyceraldehyde-3-phosphate dehydrogenase (10494), and liver kinase B1 (10746) were purchased from Proteintech (Rosemont, IL); antibodies against ARID1A (sc-32761), a-tubulin (sc-32293), b-actin (sc-47778), ubiquitin (sc-8017), and c-myc (sc-764) were purchased from Santa Cruz Biotechnology (Dallas, Texas); and antibodies against Flag and HA were purchased from Sigma-Aldrich (St. Louis, MO).

Techniques: Activity Assay, Mutagenesis, Ubiquitin Proteomics, Knock-Out, Luciferase, Reporter Assay, Expressing, Control

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: 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