Journal: International Journal of Molecular Sciences

Article Title: Inhibition of BET Proteins during Adolescence Affects Prefrontal Cortical Development: Relevance to Schizophrenia

doi: 10.3390/ijms22168710

Figure Lengend Snippet: The effects of an adolescent JQ1 administration on BRD gene expression in the adult medial prefrontal cortex in the MAM-E17 model of schizophrenia. The rats (males: A – C and females: D – F ) were exposed to JQ1 or vehicle (CON) in early adolescence (P23–P29), and the analyses were performed in rats at P110. ( A – C ) BRD2 , BRD3 , and BRD4 expression in males, respectively. ( D – F ) BRD2 , BRD3 , and BRD4 expression in females, respectively. The data are expressed as percentage of VEH-CON. Each data point represents the mean ± SEM; n = 6 per group; * p < 0.05 vs. VEH-CON, # p < 0.05 vs. MAM-CON (two-way ANOVA followed by a Tukey test).

Article Snippet: Rn01435739_m1 , BRD3 , bromodomain containing 3.

Techniques: Gene Expression, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of BET Proteins during Adolescence Affects Prefrontal Cortical Development: Relevance to Schizophrenia

doi: 10.3390/ijms22168710

Figure Lengend Snippet: A list of a gene-specific primers and probes.

Article Snippet: Rn01435739_m1 , BRD3 , bromodomain containing 3.

Techniques: Activity Assay

Journal: Frontiers in Oncology

Article Title: The MYC Paralog-PARP1 Axis as a Potential Therapeutic Target in MYC Paralog-Activated Small Cell Lung Cancer

doi: 10.3389/fonc.2020.565820

Figure Lengend Snippet: Effects of JQ1 and BMN673 on the HR repair pathway. (A) Representative images of Rad51 immunofluorescence staining in SCLC cells treated with drugs as indicated for 24 h. Scale bar, 20 μm. Quantification of Rad51 fluorescence intensities from three independent experiments was shown as mean ± S.D. **P < 0.01; ***P < 0.001. (B) Western blot analysis of Rad51 expression in SCLC cells treated with drugs as indicated for 24 h. (C) Western blot analysis of indicated proteins in DMS273 cell upon knockdown of BRD2 or BRD3 or BRD4 . (D) ChIP-PCR analysis displays the decreased association of BRD4 in the promoter of RAD51 in DMS273 and H526 cells treatment with JQ1 for 24 h. *P < 0.05; **P < 0.01; ns, no significance. (E) Western blot analysis of phosphorylated-DNA-PKcs and phosphorylated-RPA32 in MYC paralog-dependent and independent-SCLC cells treated with indicated drugs for 24 h. β-actin was used as a loading control.

Article Snippet: Antibodies against the following proteins were used for our studies: c-MYC (1:1,000, abcam ab32072), PARP1 (1:1,000, Affinity DF7198), GAPDH (1:10,000, Affinity AF7021), Rad51 (1:10,000, Abcam ab133534), PARP (1:1,000, CST 9542), γH2AX (1:1,000, CST 2577), p-CHK1 (1:1,000, Ser317, CST 12302), BRD2 (1:1,000, Proteintech 22236-1-AP), BRD3 (1:1,000, Proteintech 11859-1-AP), BRD4 (1:500, Bethyl A301-985A50), p-DNA-PKcs (1:1,000, Abcam ab124918), p-RPA32 (1:5,000, Ser4/Ser8, Novus), MYCN (1:1,000, CST 9405), MYCL (1:1,000 R&D, AF4050) and β-actin (1:10,000, Transgen HC201-02).

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Knockdown, Control

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: Melanocyte differentiation gene expression is suppressed by BET inhibition. a Melb-a cells were differentiated in the presence of vehicle (VC) or 500 nM (+)JQ1. Cells were harvested at the indicated time-points and subjected to qRT-PCR. Tyr , Tyrp1 , or Trpm1 levels were normalized to that of Rpl7 . The data are the average of three independent experiments. Standard error bars are shown. Statistically significant difference between VC and (+)JQ1 treated samples at each time point are shown (* p < 0.05, ** p < 0.01, *** p < 0.001). b Protein extracts from cells cultured in the absence (vehicle treated) or presence of (+)JQ1 were subjected to immunoblot analysis using the indicated antibodies. The figure is representative of three or more experiments. c Neonatal human epidermal melanocytes (NHEMs) were cultured in the absence (vehicle treated) or presence of 500 nM (+)JQ1 for the indicated number of days. Harvested cells were subjected to immunoblot analysis using the indicated antibodies. The figure is representative of three or more experiments. d NHEMs were cultured as in c and then subjected to qRT-PCR. The TYRP1 level was normalized to that of RPL9 . The data are the average of three independent experiments. Standard error bars are shown. Statistically significant difference between VC and (+)JQ1 treated samples at each time point are shown (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Gene Expression, Inhibition, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: Depletion of BRD4 or BRD2 in Melb-a cells suppresses expression of melanocyte-specific genes. a Melb-a cells were transfected with control siRNA or siRNA that targets BRD2, BRD3, or BRD4, then differentiated for 48 h. Immunoblot analysis was performed with the indicated antibodies. The immunoblot is representative of two or more experiments. b Melb-a cells were transfected with a control (siC) or a pool of siRNAs that targets BRD4 or BRD2 (distinct from those used in a ) then differentiated for 48 h. Immunoblot analysis was performed with the indicated antibodies. The immunoblot is representative of two or more experiments. c , d BRD4 or BRD2 depleted cells treated as described in b were subjected to qRT-PCR. Brd4 , Brd2 , Tyr , and Tyrp1 levels were normalized to that of Rpl7 . The data in c and d are the average of three independent experiments. Standard error bars are shown. Statistically significant differences between siC, siBRD4, and siBRD2 transfected cells are shown (* p < 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: BRD4 and BRD2 binding overlap H3K27ac and canonical MITF-binding sites at melanocyte-specific loci. Publicly available ChIP-seq data in normal human melanocytes, SK-MEL-5 melanoma cells (GSM1968282), 501 melanoma cells (GSM1517751), and neonatal human epidermal melanocytes (NHEM) (GSM2842798) with the indicated antibodies at the a TYR and b TYRP1 loci

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Binding Assay, ChIP-sequencing

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: BRD4 and BRD2 occupancy at MITF-binding sites is associated with chromatin accessibility in Meb-a cells. a , b Melb-a cells were differentiated for 24 h in the presence of vehicle (VC) or 500 nM (+)JQ1. Cells were cross-linked and control IgG or an antibody to BRD4 or BRD2 was used for ChIP. BRD4 and BRD2 binding to the MITF-binding sites to the promoter regions of Tyr and Tyrp1 was assayed by qPCR. BRD4 and BRD2 enrichment is shown relative to IgG enrichment and normalized to a non-specific region ( IgH enhancer). The data are the average of three independent experiments. Standard error bars are shown statistically significant differences between VC and (+)JQ1 treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001). c Melb-a cells were differentiated as described in a , b . FAIRE enrichment at the MITF-binding sites in the Tyr and Tyrp1 promoters was determined by qPCR. Enrichment at the IgH enhancer is shown as a negative control. The data are the average of two independent experiments performed in triplicate Statistically significant differences between VC and (+)JQ1-treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001). D. NHEMs were treated with 500 nM inactive (−)JQ1 or 500 nM active (+)JQ1 for 48 h, then harvested for FAIRE. FAIRE enrichment at the MITF-binding site in the TYRP1 promoter was determined by qPCR. FAIRE enrichment at the CD25 promoter is shown as a negative control. The data is the average of two independent experiments performed in triplicate Statistically significant differences between (−)JQ1 and (+)JQ1-treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Binding Assay, Control, Negative Control

Journal: Epigenetics & Chromatin

Article Title: Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation

doi: 10.1186/s13072-020-00333-z

Figure Lengend Snippet: BRD4 and BRD2 promote MITF occupancy at the Tyr and Tyrp1 promoters. a Melb-a cells were differentiated and cross-linked as described in Fig. . ChIPs were performed with a control IgG or an antibody to MITF. MITF enrichment is shown relative to IgG enrichment and normalized to a non-specific region ( IgH enhancer). The data are the average of three independent experiments. Standard error bars are shown. Statistically significant differences between (−)JQ1 and (+)JQ1 treated samples are shown (*< 0.05, ** p < 0.01, *** p < 0.001). b Melb-a cells were transfected with control siRNA (siC) or a pool of siRNAs that target either BRD4 or BRD2. Cells were differentiated for 24 h then harvested for ChIPs. MITF enrichment is shown relative to IgG enrichment and normalized to a non-specific region ( IgH enhancer). The data are the average of two independent experiments performed in triplicate. Standard error bars are shown. Statistically significant differences between siC-transfected cells and siBRD4 and siBRD2-transfected cells are shown (*< 0.05, ** p < 0.01, *** p < 0.001)

Article Snippet: The BRD3 (sc-81202), TYR (sc-7833), and TYRP1 (sc-10443) antibodies were from Santa Cruz Technologies (Santa Cruz, CA, USA).

Techniques: Control, Transfection

Journal: Biomolecules

Article Title: The Inhibition of Bromodomain and Extraterminal Domain (BET) Proteins Protects Against Microglia-Mediated Neuronal Loss In Vitro

doi: 10.3390/biom15040528

Figure Lengend Snippet: The effect of LPS on BET protein expression in BV2 microglial cells. BV2 cells were treated with LPS (100 ng/mL) for 6 h. ( a ) mRNA levels of Brd2 , Brd3 , and Brd4 were analyzed using qPCR. ( b ) Protein levels of BET proteins were measured using ELISA assays. Data are presented as mean ± SEM; n = 4 ( a ) and 3–4 ( b ); * p < 0.05 and ** p < 0.01, compared to the control (Student’s t -test).

Article Snippet: The levels of mRNA for selected genes were quantified using TaqMan Gene Expression Assays, including Arg1 (Mm00475988_m1), Brd2 (Mm01271171_g1), Brd3 (Mm01326697_m1), Brd4 (Mm01350417_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Nos2 (Mm00440502_m1), Tnf (Mm00443258_m1), and Gusb (Mm01197698_m1) [ ].

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: Silencing of BRD3 in FLS. The expression of BRD3 was silenced in FLS from wrist joints by transduction of lentiviral particles prior to stimulation with TNF (10 ng/mL). Transcriptomes were analyzed by RNAseq. ( a ) Expression levels of BRD3 , BRD2 , and BRD4 after silencing of BRD3 in RNAseq data sets. ( b ) Silencing of BRD3 was confirmed by Western blotting. Volcano plots of RNAseq data sets in ( c ) unstimulated and ( d ) TNF-stimulated FLS after silencing of BRD3. ( e ) Venn diagram of BRD3-regulated genes (±fold change > 1.5; FDR ≤ 0.1) in unstimulated and TNF-stimulated FLS. * p < 0.05.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Transduction, Western Blot

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: Pathway enrichment analysis of FLS silenced for BRD3. Differentially expressed genes (±fold change > 1.5; FDR ≤ 0.1) of FLS silenced for BRD3 entered pathway enrichment analysis. ( a ) Reactome pathways shared between unstimulated and TNF-stimulated FLS are shown. The full list of enriched pathways, and genes enriched in these pathways, can be found in . cnet plots depicting the linkages of genes in enriched pathways ( b ) in unstimulated and ( c ) TNF-stimulated FLS.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques:

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: Expression of BET proteins in subtypes of FLS. ( a ) Visualization of different subtypes of FLS by UMAP using scRNA-seq data sets available at the BroadSingleCellPortal . ( b ) BRD2 , BRD3 , and BRD4 expression in subtypes of FLS. ( c ) Dot plots indicating BRD2 , BRD3 , and BRD4 expression in synovial tissues with different inflammatory scores as defined by inflammatory cell infiltration. Correlation of ( d ) mean BRD3 expression and ( e ) percentage of BRD3 -positive cells with inflammatory scores of synovial tissues.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: Inflammatory target genes of BRD3. FLS from hand (circles) and shoulder (squares) were silenced for BRD3 and stimulated with TNF or left untreated. The expression of inflammatory genes, identified to be differentially expressed by RNAseq, was measured by real-time PCR. * p < 0.05.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: BRD3-regulated stress response in FLS. ( a ) FLS from hand (circles) and shoulder (squares) were silenced for BRD3 and stimulated with TNF or left untreated. The expression of stress response-related genes was measured by real-time PCR. FLS from hand (circles) and shoulder (squares) were treated with I-BET in the absence and presence of TNF. ( b ) VEGF secretion and ( c ) extracellular lactate levels in cell culture supernatants. * p < 0.05; ** p < 0.01.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: Oxidative stress regulates the expression of BRD3 in FLS. FLS from hand (circles) and shoulder (squares) were stimulated with 4-HNE in the absence and presence of TNF. The expression of BRD3 was measured by Western blotting. ( a ) A representative Western blot is shown. ( b ) Densitometric analysis of Western blot results. * p < 0.05.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot

Journal: Biomedicines

Article Title: BRD3 Regulates the Inflammatory and Stress Response in Rheumatoid Arthritis Synovial Fibroblasts

doi: 10.3390/biomedicines11123188

Figure Lengend Snippet: I-BET induces autophagy in FLS. ( a ) Heatmap of autophagy-related genes identified by RNAseq in FLS silenced for BRD3. ( b ) FLS from hand (circles) and shoulder (squares) joints were treated with I-BET in the absence and presence of bafilomycin (Baf). The conversion of LC3B-I to LC3B-II and the expression of p62 and α-tubulin were measured by Western blotting. A representative Western blot is shown. Densitometric analysis of Western blot results for ( c ) levels of LC3B-II and ( d ) p62. ( e ) Formation of autophagosomes in I-BET-treated FLS ( n = 6) was measured by live cell imaging in the absence and presence of chloroquine (CQ). Representative images for each condition (in shoulder FLS) are shown. ( f ) Average spot areas and ( g ) average spot counts were calculated from quadruplicates for each condition and patient sample. * p < 0.05; ** p < 0.01; *** p < 0.005.

Article Snippet: FLS were transduced with lentiviral particles targeting BRD3 or control particles (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot, Live Cell Imaging