Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Continuous Activation of C/EBPβ Transcription Factor Prevents Fibrosis Resolution After Alcohol Cessation

doi: 10.1016/j.jcmgh.2025.101525

Figure Lengend Snippet: C/EBPβ inhibition promotes proresolving phenotype in liver macrophages. ( A ) WT or Cebpb KO hepatocytes were cocultured with WT liver macrophages in a Transwell coculture system. Macrophage collagen degradation activity, n = 4 per group. ∗∗ P < .01. ( B ) RNA-seq data of Cyp3a gene expression fold change in WT mice during resolution (Res/WDA) and Cebpb KO mice during resolution (KO_Res/WDA). ( C ) Relative mRNA of Cyp3a16 and Cyp3a41 ( Cyp3a41a+ Cyp3a41b ) in WT and KO mice at 4 weeks after stopping alcohol. ( D ) Schematic of Cyp3a gene cluster showing ATAC peaks position and gRNA position. ( Bottom ) Differentially accessible peaks in hepatocytes from resolution condition compared with control hepatocytes. ( E ) Primary hepatocytes were transfected with dCas9-p300 vector and single gRNA targeting Cyp3a region or scrambled control. Relative mRNA expression in hepatocytes. ( F ) After 48 hours of incubation hepatocyte conditioned media was used for HDL isolation. Liver macrophages were treated with isolated HDL from hepatocytes expressing scr control RNA or Cyp3a targeting RNA. Relative mRNA in macrophages, n = 4–8 per group. ∗ P < .05; ∗∗ P < .01. ( G ) Macrophages were tested in a collagen degradation assay. n = 5 per group. ∗ P < .05. ( H ) Macrophages were treated with 10 μM of corresponding oxysterols. ( Right ) Relative mRNA in macrophages, n = 4–8 per group. ∗∗ P < .01. ( Right ) Whole liver LXRα target gene expression log2Fold (KO/WT). ( Bottom ) Correlation between CYP3A7 and LRXα target genes in human liver (TCGA + GTEx) samples. ( I ) Macrophages were treated with 10 μM of corresponding oxysterols. After 24 hours of culture macrophages were tested in a collagen degradation assay. ( J ) Relative mRNA in macrophages, n = 4–8 per group. ∗ P < .05; ∗∗ P < .01. ( K ) Correlation between CYP3A7 and proresolving macrophage marker genes in human liver (TCGA + GTEx) samples.

Article Snippet: AAV-TBG-control and AAV-TBG-iCre were from VectorBiolabs (Malvern, PA) and were used at 1 x 10 11 genome copies per mouse (Cre/control). dCas9-p300 plasmid was from Addgene (pcDNA-dCas9-p300 Core, Cat# 61357).

Techniques: Inhibition, Activity Assay, RNA Sequencing, Gene Expression, Control, Transfection, Plasmid Preparation, Expressing, Incubation, Isolation, Degradation Assay, Targeted Gene Expression, Marker

Journal: Cell Death & Disease

Article Title: FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

doi: 10.1038/cddis.2013.109

Figure Lengend Snippet: FKBP51 interacts with the general transcriptional co-activator p300. ( a , left) FKBP51 co-immunoprecipitates with p300. ( a , right), p300 co-immunoprecipitates with FKBP51. Total-cell lysates were prepared by SAN melanoma cells transfected with FKBP51/Flag. Cell lysates were immunoprecipitated with anti-Kat3B/p300 (IP p300) or anti-Flag (IP FKBP51). Immunoprecipitated and total lysates were then subjected to western blot with anti-FKBP51 or anti-p300. ( b ) ChIP performed with SAN melanoma cells, silenced (FKBP51 siRNA) or not (NS RNA) for FKBP51. An enrichment of DNA (region at −3450 from TSS of ABCG2 gene) can be observed in p300-immunoprecipitated chromatin (NS RNA) compared with IgG sample. Such an enrichment appeared to be reduced when FKBP51 was silenced (FKBP51 siRNA)

Article Snippet: The p300- immunoprecipitated chromatin was obtained using KAT3B/p300 Antibody (RW109) (Novus Biologicals).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: Prothymosin α overexpression contributes to the development of pulmonary emphysema

doi: 10.1038/ncomms2906

Figure Lengend Snippet: ( a , b ) Immunohistochemical staining for HDACs. Lung tissues from patients with emphysema of varying severity and non-COPD individuals ( a ), as well as lung tissues from 4-day-old ProT HZ, HET and NT mice ( b ) were immunostained with monoclonal antibody against HDAC1, HDAC2 and HDAC3. Note that lower levels of HDAC2, but not HDAC1 and HDAC3, were detected in patients with more severe emphysema and in ProT HZ mice, as compared with their normal counterparts. The boxed areas in upper panels (original magnification × 400; scale bar=100 μm) are magnified and shown in lower panels. ( c ) Immunoblot analysis of HDACs in the lung tissues of 4-day-old ProT HZ, HET and NT mice. ( d ) Detection of acetylated-lysine (acetyl-K). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviral vectors expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression plasmid (pCMVb p300 HA tag) or a control plasmid (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression were verified. ( e ) Detection of acetylated histones. 293 T cells were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 and their total cell lysates were examined for the indicated proteins by immunoblotting after 24 h. ( f ) HDAC and histone binding assay. 293T cells that had been transduced with HDAC1-Flag (pcDNA3.1-HDAC1-Flag) (left) or HDAC2-Flag (pCMV-Tag4C-HDAC2) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. Total cell lysates were immunoprecipitated by Flag-M2 beads and analysed by immunoblotting for the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of histone H1 and H3, HDAC1, HDAC2 and ProT by immunoblotting. ( g ) Proposed model for ProT-mediated enhancement of histone acetylation via HDAC/histone dissociation. Results are representative of three independent experiments.

Article Snippet: The commercially available plasmids that were used in this study included pCMVb p300 HA delta33, which encodes human p300 mutant protein lacking amino acids 1737–1836; pcDNA3.1-HDAC1-Flag; pcDNA3.1-HDAC3-Flag; and pcDNA3-cFlag-RelA, each of which was purchased from Addgene; as well as pHM6-lacZ (Roche), pNF-κB-Luc (Clontech), pTK-Renilla (Promega) and pCMV-Tag2B (Stratagen).

Techniques: Immunohistochemical staining, Staining, Western Blot, Transfection, Transduction, Expressing, shRNA, Plasmid Preparation, Control, Binding Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: Prothymosin α overexpression contributes to the development of pulmonary emphysema

doi: 10.1038/ncomms2906

Figure Lengend Snippet: ( a ) Detection of acetylated NF-κB p65 (Lys310). 293T cells that had been transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1 (left) or transduced with lentiviruses expressing ProT shRNA-1 and -2 or GFP shRNA (right) were transfected with a p300 expression vector (pCMVb p300 HA delta33) or a control vector (pHM6-lacZ). After 24 h, total cell lysates were examined for the indicated proteins by immunoblotting. Equal levels of p300 and LacZ expression in the transfected cells were verified. ( b ) Reporter assay for NF-κB transactivation activity. 293T cells that had been transduced with lentiviruses expressing ProT-IRES-GFP or GFP alone (left), or ProT or GFP shRNA (right) were cotransfected with pNF-κB-Luc and pTK-Renilla reporter plasmids. Total cell lysates were harvested 48 h later, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light activity. Values shown are the mean±s.e.m. ( n =4 for left and n =5 for right; Student’s t -test). ( c ) NF-κB and ProT binding assay. Cell lysates from 293T cells transduced with lentiviruses expressing GFP-tagged ProT or GFP alone were immuoprecipitated by anti-GFP monoclonal antibody followed by immunoblotting with anti-NF-κB p65 and anti-GFP antibody. Whole-cell lysates without immunoprecipitation (10% input) were estimated for the expression levels of NF-κB and ProT by immunoblotting. ( d ) Analysis of the effect of ProT on NF-κB and HDAC3 binding. 293T cells that had been transfected with an NF-κB p65 expression vector (pcDNA3-cFlag-RelA) or a control vector (pCMV-Tag2B) (left) or HDAC3 expression vector (pcDNA3.1-HDAC3-Flag) or a control vector (pCMV-Tag2B) (right) were transfected with pcDNA3.1-ProT-myc/His or pcDNA3.1. After 24 h, total cell lysates were immuoprecipitated by Flag-M2 beads followed by immunoblotting with the indicated proteins. Whole-cell lysates without immunoprecipitation (10% input) were estimated by immunoblotting with indicated proteins. ( e ) p300 and NF-κB binding assay. 293T cells that had been transduced with lentiviruses expressing ProT shRNA-2 or luciferase shRNA as the control were transfected with the pCMVb p300 HA delta33 and pcDNA3-cFlag-RelA. Total cell lysates were immunoprecipitated by anti-HA antibody followed by immunoblotting with the indicated proteins. Results are representative of three independent experiments. ( f ) Proposed model for ProT-mediated NF-κB hyperacetylation via HDAC3/NF-κB dissociation and p300/NF-κB association.

Article Snippet: The commercially available plasmids that were used in this study included pCMVb p300 HA delta33, which encodes human p300 mutant protein lacking amino acids 1737–1836; pcDNA3.1-HDAC1-Flag; pcDNA3.1-HDAC3-Flag; and pcDNA3-cFlag-RelA, each of which was purchased from Addgene; as well as pHM6-lacZ (Roche), pNF-κB-Luc (Clontech), pTK-Renilla (Promega) and pCMV-Tag2B (Stratagen).

Techniques: Transfection, Transduction, Expressing, shRNA, Plasmid Preparation, Control, Western Blot, Reporter Assay, Activity Assay, Luciferase, Binding Assay, Immunoprecipitation