ihc staining of brd4  (Human Protein Atlas)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The BRD4 inhibitor JQ1 augments the antitumor efficacy of abemaciclib in preclinical models of gastric carcinoma

doi: 10.1186/s13046-023-02615-2

Figure Lengend Snippet: JQ1 is a promising candidate in combination with ABE. A Ranked synergy models of ABE and screened epigenetic drugs, including JQ1, tazemetostat, vorinostat, 5-azacitidine, SETDB1-TTD-IN-1 and UNC669. Synergy scores were labelled to the top of each model. B Dependency score of CDK4 and BRD4 for tumor cells across different cancer types. Score < 0 represents essential genes and Score < -1 represents super essential genes. C Transcriptional expression level of BRD4 between tumor and normal tissues in TCGA-STAD cohort. D Immunohistochemistry (IHC) staining of BRD4 in GC patients from The Human Protein Atlas. E CUT&Tag-seq binding enrichment of H3K27ac in AGS cells treated with DMSO or ABE. F Pie plot of the genomic distribution of H3K27ac peaks in ABE-treated AGS cells. G Gene tracks depicting H3K27ac signals at the BRD4 locus. The signal from ABE and DMSO was normalized at same level. H Western blotting images showing the protein level of BRD4 protein level after ABE treatment. GAPDH was used as a loading control. I Quantification of the protein level of BRD4 in AGS cells after ABE treatment for 24 h using ImageJ software. The data are presented as the mean ± SEM of three replicates. J Enrichment analysis of top1000 upregulated H3K27ac peaks in ABE-treated AGS cells. K Gene tracks depicting H3K27ac signals at the SMAD3, VCL, SNAI1 and RAC1 locus. The signals from ABE and DMSO were normalized at same level

Article Snippet: D Immunohistochemistry (IHC) staining of BRD4 in GC patients from The Human Protein Atlas.

Techniques: Expressing, Immunohistochemistry, Binding Assay, Western Blot, Control, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The BRD4 inhibitor JQ1 augments the antitumor efficacy of abemaciclib in preclinical models of gastric carcinoma

doi: 10.1186/s13046-023-02615-2

Figure Lengend Snippet: CDK4/6 and BRD4 Inhibition Specifically induce cell senescence and DNA damage in vitro. A , C , E Immunofluorescence staining of the DNA damage marker γH2AX (scale bar = 25 μm), DNA senescence marker p21 and p53 (scale bar = 50 μm ) in AGS cell line upon the treatment of the indicted agents for 24 h. B , D , F Quantification of the positive cell ratio and mean fluorescence intensity using QuPath software. The data are presented as the mean ± SEM of three replicates. G Western blotting images showing the protein level of Phospho-Rb (Ser807/811), γH2AX, p53 and p21 in AGS cells from the indicted treatment cohort. GAPDH was used as a loading control. H Quantification of the protein level of Phospho-Rb (Ser807/811), γH2AX, p53 and p21 in AGS cells from the indicted treatment for 24 h using imageJ software. The data are presented as the mean ± SEM of three replicates. Significance of each group was compared with DMSO group

Article Snippet: D Immunohistochemistry (IHC) staining of BRD4 in GC patients from The Human Protein Atlas.

Techniques: Inhibition, In Vitro, Immunofluorescence, Staining, Marker, Fluorescence, Software, Western Blot, Control

Journal: Cell Death & Disease

Article Title: BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis

doi: 10.1038/s41419-017-0181-6

Figure Lengend Snippet: a Box-plots of BRD4 mRNA levels in normal tissue (NT, patient number = 35) vs primary of tumor (TP, patient number = 415) acquired from TCGA Firebrowse portal are visualized using GraphPad Prism, and the p -value is computed and displayed. b Left: Representative of immunohistochemical (IHC) staining of BRD4 in human NT vs TP tissues. Boxed regions are enlarged to the bottom of each image. Right: IHC staining score summary from 52 gastric cancer samples and the paired normal tissue samples is shown in the table on the right. The Pearson’s chi-square test ( χ 2 = 68.250, p < 0.001) is utilized to evaluate the likelihood of the different expression levels of BRD4 in NT vs TP samples. c Various gastric cancer cells were treated with JQ1 of indicated concentration for 72 h, and cell proliferation was measured by A490 nm using the CellTiter 96 R AQueous One Solution cell proliferation assay (MTS) (Promega). Data represent the mean of three independent experiments. Dot line represents the 50% of growth inhibition. d MKN28 or SGC-7901 cells were treated with DMSO or 5 μM of JQ1 for 24 h or 6 days, and cell proliferation was measured at different time points as in c . e MKN28 cells were seeded in soft-agar and cultured for 15 days with DMSO or 5 μM of JQ1. Representative photographs were taken at day 21. f MKN28 cells were treated with DMSO or 5 μM of JQ1, and cell invasion assay was performed using Transwell invasion chambers (Becton, Dickinson, and Company) according to manufacturer’s instructions. g The wound-healing migration assays for MKN28 cells in the presence of DMSO or 5 μM of JQ1. Representative photographs were taken at 0, 18, and 36 h (left). The percentage of the average speed of wound closure from three independent experiments ± SD is shown on the right

Article Snippet: Fig. 1 JQ1 inhibits the proliferation, migration, and invasion of gastric cancer cell lines. a Box-plots of BRD4 mRNA levels in normal tissue (NT, patient number = 35) vs primary of tumor (TP, patient number = 415) acquired from TCGA Firebrowse portal are visualized using GraphPad Prism, and the p -value is computed and displayed. b Left: Representative of immunohistochemical (IHC) staining of BRD4 in human NT vs TP tissues.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Concentration Assay, Proliferation Assay, Inhibition, Cell Culture, Invasion Assay, Migration

Journal: Cell Death & Disease

Article Title: BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis

doi: 10.1038/s41419-017-0181-6

Figure Lengend Snippet: BRD4 is involved in JQ1-induced cellular senescence . a MKN28 cells were infected with lentiviruses expressing shRNAs against Brd2 , Brd3 , and Brd4 , respectively. Cell proliferation was measured by clonogenic assay after 3 days. Knockdown efficiency is shown on the right panels. b MKN28 cells infected with lentiviruses expressing indicated shRNAs were subject to β-Gal staining as described in Fig. . Percentage of β-Gal staining-positive cells is shown on the right. c MKN28 cells infected with lentiviruses expressing indicated shRNAs were lysed and subject to immunoblotting for indicated proteins. d&e MKN28 cells were transfected with either control or p21 siRNA. Twenty hours later, cells were treated with DMSO or 5 μM of JQ1 for another 3 days. β-Gal staining was performed as in Fig. . Percentage of β-Gal staining-positive cells is shown on the right. Data represent the average of three independent experiments. p21 siRNA knockdown efficiency is shown in ( e )

Article Snippet: Fig. 1 JQ1 inhibits the proliferation, migration, and invasion of gastric cancer cell lines. a Box-plots of BRD4 mRNA levels in normal tissue (NT, patient number = 35) vs primary of tumor (TP, patient number = 415) acquired from TCGA Firebrowse portal are visualized using GraphPad Prism, and the p -value is computed and displayed. b Left: Representative of immunohistochemical (IHC) staining of BRD4 in human NT vs TP tissues.

Techniques: Infection, Expressing, Clonogenic Assay, Knockdown, Staining, Western Blot, Transfection, Control

Journal: Cell Death & Disease

Article Title: BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis

doi: 10.1038/s41419-017-0181-6

Figure Lengend Snippet: a Scatterplots of Brd4 mRNA expression level versus p21 / CDKN1A mRNA expression level in Stomach Adenocarcinoma (STAD) in The Cancer Genome Atlas (TCGA). Data are acquired from TCGA cBioportal and analyzed by R programming. 95% Confident Intervals (CI), Pearson correlation coefficients and P values are displayed. b MKN28 cells were transfected with control siRNA, and two sets of BRD4 siRNA for 24 h and the levels of Brd4 and p21 mRNA were measured by RT-PCR. Data represent the average of three independent experiments. c MKN28 cells were treated with DMSO or different concentrations of JQ1 for 24 h and the levels of p21 mRNA were measured by RT-PCR. Data represent the average of three independent experiments. d The p21 3′-UTR luciferase reporter plasmids were transfected into MKN28 cells with or without JQ1 treatment. Luciferase activity was measured as indicated time points after transfection. Data represent the average of three independent experiments. e MKN28 cells were infected with lentiviruses expressing control or Brd4 shRNA. 24 h later, infected cells were transfected with p21 3′-UTR luciferase reporter plasmids. Luciferase activity was measured 24 h after transfection. Data represent the average of three independent experiments. f MKN28 cells were transfected with p21 3′-UTR luciferase reporter plasmids together with expression vectors for BRD4 or BRD4(ΔBDs). Luciferase activity was measured 48 h after transfection. Data represent the average of three independent experiments

Article Snippet: Fig. 1 JQ1 inhibits the proliferation, migration, and invasion of gastric cancer cell lines. a Box-plots of BRD4 mRNA levels in normal tissue (NT, patient number = 35) vs primary of tumor (TP, patient number = 415) acquired from TCGA Firebrowse portal are visualized using GraphPad Prism, and the p -value is computed and displayed. b Left: Representative of immunohistochemical (IHC) staining of BRD4 in human NT vs TP tissues.

Techniques: Expressing, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Infection, shRNA

Journal: Cell Death & Disease

Article Title: BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis

doi: 10.1038/s41419-017-0181-6

Figure Lengend Snippet: a Schema of the miRNA candidates targeting p21 mRNA. The top two circles represent the number of predicted p21 miRNAs using prediction algorithms Target Scan (left) and miRDB (right). The bottom circle represents the number of miRNAs that are up-regulated in gastric cancer tissues compared to normal tissues. b MKN28 cells were transfected with control or BRD4 siRNA for 24 h. The levels of indicated miRNAs were measured using QuantiMir Kit (System Bioscience). Samples are from si BRD4-1 in Fig. . c MKN28 cells transfected with indicated miRNA mimics for 24 h were treated with JQ1 for 24 h and the levels of p21 were measured by immunoblotting with anti-p21 antibody. d Sequence complementarity between miR-106b-5p and 3′-UTR of p21 mRNA (top panel). miR-106b-5p mimics and inhibitors were transfected into MKN28 cells. Forty-eight hours later, the levels of indicated proteins were measured by immunoblotting (bottom panel). e MKN28 cells were transfected with miR-106b-5p inhibitors for 5 days, and cells were subject to β-Gal staining (left panel). The percentage of β-Gal staining-positive cells is indicated in the right. f MKN28 cells were transfected with different combinations of BRD4 siRNA and miR-106b-5p mimics as indicated. After 5 days, cells were subject to β-Gal staining. The percentage of β-Gal staining-positive cells is indicated on the right

Article Snippet: Fig. 1 JQ1 inhibits the proliferation, migration, and invasion of gastric cancer cell lines. a Box-plots of BRD4 mRNA levels in normal tissue (NT, patient number = 35) vs primary of tumor (TP, patient number = 415) acquired from TCGA Firebrowse portal are visualized using GraphPad Prism, and the p -value is computed and displayed. b Left: Representative of immunohistochemical (IHC) staining of BRD4 in human NT vs TP tissues.

Techniques: Transfection, Control, Western Blot, Sequencing, Staining

Journal: Cell Death & Disease

Article Title: BRD4 regulates cellular senescence in gastric cancer cells via E2F/miR-106b/p21 axis

doi: 10.1038/s41419-017-0181-6

Figure Lengend Snippet: a UCSC genome browser display of MCM7 gene which hosts miR-106b-5p on Chr.7 (GRCh38/hg38) assembly. Light box: intron; dark box: exon. b and c MKN28 cells were treated with either JQ (5 μM) (B) or HLM006474 (20 μM) (C) for 24 h. ChIP assay was performed using antibodies against IgG or BRD4 and probed for the promoter region of miR-106b-5p. d MKN28 cells were treated with HLM006474 (20 μM) for 24 h and the levels of MCM7 mRNA and mature miR-106b-5p were measured by RT-PCR. e MKN28 cells were treated with HLM006474 (10 μM and 20 μM) for 24 h and the levels of p21 and cyclin B1 were measured by immunoblotting. f MKN28 cells were treated with indicated concentration of HLM006474 for 4 days and the cells were subject to β-Gal staining. The percentage of β-Gal staining-positive cells is shown on the right. Data represent the average of three independent experiments. g Schematic model for the regulation of p21 and cellular senescence by BRD4 in gastric cancer cells. In cancer cells, BRD4 is recruited to the promoter of miR-106b-5p via E2F proteins and activates the expression of miR-106b-5p, which targets the 3′-UTR of p21 mRNA and suppresses its expression to promote cell proliferation. Inhibition of BRD4 by siRNA or JQ1 results in the down-regulation of miR-106b-5p, leading to the increased expression of p21 and cellular senescence

Article Snippet: Fig. 1 JQ1 inhibits the proliferation, migration, and invasion of gastric cancer cell lines. a Box-plots of BRD4 mRNA levels in normal tissue (NT, patient number = 35) vs primary of tumor (TP, patient number = 415) acquired from TCGA Firebrowse portal are visualized using GraphPad Prism, and the p -value is computed and displayed. b Left: Representative of immunohistochemical (IHC) staining of BRD4 in human NT vs TP tissues.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Concentration Assay, Staining, Expressing, Inhibition