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: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , b , Structure ( a ) and BET protein degradation activity ( b ) of sulfonamide-based PROTAC DAT389. HeLa cells were treated with the indicated concentrations of MZ1 or DAT389 for 16 hours and BET protein levels were analysed by immunoblot. c , d , Structures ( c ) and degradation activities ( d ) of compounds 1 and 2 . BRD2 and BRD4(S) protein remaining after compound treatment in HEK293, HCT-116 WT and DCAF15 KO cells based on immunoblot quantification. Source data, Supplementary Fig. 1a. e, Cytotoxicity of compounds 1 and 2 . MV4;11 and HCT-116 cells were treated with increasing concentrations of 1 and 2 for 24 or 96 hours, respectively, and cell viability was assessed via CellTiterGlo assay. Dose-response curves were fitted using non-linear regression fits. Data shown are from n = 2 biological repeats, mean +/- s.d. f , In-cell mechanistic evaluation of 1 . HCT-116 cells were treated for 2 hours with E7820 (1 µM) or 1 (10 nM) alone, or after 1 hour pre-treatment with JQ1 (10 µM), MG132 (50 µM) or MLN4924 (3 µM). Western blot representative of n = 3 independent experiments. g, NanoBRET kinetic degradation assay. BromoTag-HiBiT-BRD4 knock-in HEK293 cells were treated with compound 1 at indicated concentrations with or without MLN4924 (10 µM) 1-hour pre-treatment. n = 3 biological repeats. h, DCAF15 independent protein degradation. HCT-116 WT and DCAF15 KO cells were treated with increasing concentrations of compound 2 for 6 h and BET protein levels were assessed via immunoblot. Western blots representative of n = 3 independent experiments. i , In-cell mechanistic evaluation of 1 . HCT-116 DCAF15 knockdown cells were treated for 2 hours with E7820 (1 µM) or 1 (10 nM) alone, or after 1 hour pre-treatment with JQ1 (10 µM), MG132 (50 µM) or MLN4924 (3 µM). Western blots representative of n = 2 independent experiments. j , alphaLISA displacement assay with biotinylated JQ1 probe and His-BRD4 BD2 . Data shown are from n = 3 technical replicates, mean +/- s.d. k , End-point HiBiT protein degradation. HEK293 cells were engineered using CRISPR knock-in of the HiBiT tag at the endogenous BRD2, BRD3 or BRD4 loci, respectively, and were treated with the indicated compounds for 5 h. Levels of HiBiT-tagged proteins were detected via luminescence using the HiBiT lytic detection system. Dose-response curves were fitted using non-linear regression and the data shown are form n = 3 independent repeats, mean +/- s.d.

Article Snippet: For the engineering of the fluorescent protein stability reporters, BRD4(S) (Twist Bioscience), BRD2 (Addgene plasmid # 65376) or BRD3 (Addgene plasmid # 65377; both gifts from Kyle Miller ) was cloned into a pRRL lentiviral vector, fused to 3x V5-tags and mTagBFP, and coupled to mCherry for normalization.

Techniques: Activity Assay, Western Blot, Degradation Assay, Knock-In, Knockdown, CRISPR

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , Size exclusion chromatography (SEC) UV chromatogram. DCAF16:DDB1ΔBPB:DDA1 or BRD4 Tandem in the with or without excess compound 1 were run on an S200 10/300 column as in , b. b , Schematic representation of alphaLISA displacement assay in . His-tagged BRD4 Tandem is mixed with a biotinylated-JQ1 probe. Binding of the probe to BRD4 bromodomains in the presence of nickel-chelate acceptor beads and streptavidin-europium donor beads induces proximity of donor and acceptor, energy transfer and light emission (left). Competition of the probe from BRD4 Tandem by compound 1 disrupts donor-acceptor proximity and energy transfer, and the presence of DCAF16 further strengthens compound 1 binding over probe binding (positive cooperativity; α > 1; right). c - f , Protein stability reporter assay. WT or truncated forms of BRD2, BRD3 or BRD4 ( c , e ) fused to mTagBFP were stably expressed in KBM7 cells and protein stability after 6-hour treatment with DMSO, GNE-0011 (1 µM), compound 1 (1 nM) or dBET6 (10 nM) was quantified via flow cytometric evaluation of the mTagBFP/mCherry ratio ( d , f ) as in . BD, bromodomain; NPS, N-terminal phosphorylation sites; BID, basic residue-enriched interaction domain; ET, extraterminal domain; SEED, Serine/Glutamic acid/Aspartic acid-rich region; NLS, Nuclear localization signal. Data shown are from n = 3 biological replicates, mean +/- s.d. g , h , BromoTag degradation. HEK293 cells stably expressing BromoTag-MCM4 were treated for 5 hours with DMSO, BromoTag degrader AGB1 and non-degrader cis-AGB1 , compound 1 , or ‘bumped’ analogue compound 5 ( g ) and BromoTag-MCM4 levels were analysed by immunoblotting ( h ). Data representative of n = 2 replicates.

Article Snippet: For the engineering of the fluorescent protein stability reporters, BRD4(S) (Twist Bioscience), BRD2 (Addgene plasmid # 65376) or BRD3 (Addgene plasmid # 65377; both gifts from Kyle Miller ) was cloned into a pRRL lentiviral vector, fused to 3x V5-tags and mTagBFP, and coupled to mCherry for normalization.

Techniques: Size-exclusion Chromatography, Binding Assay, Reporter Assay, Stable Transfection, Phospho-proteomics, Residue, Expressing, Western Blot

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , b , Size exclusion chromatography (SEC) UV chromatograms. DCAF16:DDB1ΔBPB:DDA1 and BRD4 Tandem alone or mixed at a 2:1 molar ratio in the presence of excess compound 1 ( a ), DCAF16:DDB1ΔBPB:DDA1 and BRD4 Tandem mixed at a 1:1 molar ratio in the absence or presence of excess compound 1 ( b ), as well as DCAF16:DDB1ΔBPB:DDA1 mixed with BRD4 BD1 and BRD4 BD2 at a molar ratio of 1:1:1 with excess compound 1 ( b ) were run on an S200 10/300 column. c , alphaLISA displacement assay. Competition of a biotinylated-JQ1 probe by compound 1 with His-BRD4 Tandem or His-BRD4 BD1 in the presence or absence of DCAF16. Data shown are from n = 2 independent experiments each with 3 technical replicates, mean +/- s.d. d , TR-FRET proximity assay. 200 nM His-tagged BRD4 (BD1, BD2 or Tandem), bound to anti-His europium acceptor beads, were incubated with increasing concentrations of Cy5-labelled DCAF16:DDB1ΔBPB:DDA1 complex and 1 µM compound 1 or DMSO. Data shown are from n = 2 independent experiments each with 2 technical replicates, mean +/- s.d. e - h , BRD4 protein stability reporter assay. BRD4(S) as well as BET protein truncations or mutants ( e , g ) fused to mTagBFP were ectopically expressed in KBM7 cells and protein stability after 6-hour treatment with DMSO, GNE-0011 (1 µM), compound 1 (1 nM) or dBET6 (10 nM) was quantified via flow cytometric evaluation of the mTagBFP/mCherry ratio ( f , h ). BD, bromodomain; NPS, N-terminal phosphorylation sites; BID, basic residue-enriched interaction domain; ET, extraterminal domain; SEED, Serine/Glutamic acid/Aspartic acid-rich region. Data shown are from n = 3 biological replicates, mean +/- s.d. i , Structural overlay of BRD2 BD2 , BRD3 BD2 and BRD4 BD2 , PDB codes 3ONI, 2OO1 and 6C7Q, respectively, along with JQ1 in the acetylated lysine binding pocket of BRD2 BD2 . Residue E344 of BRD3, shown in stick representation, is a glycine residue at the equivalent position in BRD2 and BRD4.

Article Snippet: For the engineering of the fluorescent protein stability reporters, BRD4(S) (Twist Bioscience), BRD2 (Addgene plasmid # 65376) or BRD3 (Addgene plasmid # 65377; both gifts from Kyle Miller ) was cloned into a pRRL lentiviral vector, fused to 3x V5-tags and mTagBFP, and coupled to mCherry for normalization.

Techniques: Size-exclusion Chromatography, Proximity Assay, Incubation, Reporter Assay, Phospho-proteomics, Residue, Binding Assay

Journal: Molecular cell

Article Title: Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting

doi: 10.1016/j.molcel.2024.01.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-BRD3 antibody , Santa Cruz , Cat#sc81202; RRID: AB_1119692.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Protease Inhibitor, Sequencing, Gel Extraction, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Electron Microscopy, Homologous Recombination, CRISPR, Software, Expressing, Mass Spectrometry

Journal: Frontiers in Oncology

Article Title: Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells

doi: 10.3389/fonc.2019.01471

Figure Lengend Snippet: BETd-260 induces degradation of BRD2, BRD3, and BRD4 in HCC cells. (A) HepG2 cell line was treated by BETd-260, HJB-97, or JQ1 as indicated for 24 h. The protein levels of BRD2, BRD3 and BRD4 were examined by western blot analysis. Actin was used as a loading control. (B) HepG2 cell line was treated by BETd-260 at 100 nmol/L for different times. The protein levels of BRD2, BRD3, and BRD4 were examined by western blot analysis. Actin was used as a loading control. (C) BEL-7402, SK-HEP-1, SMMC-7721, HuH-7, and MHCC97H cell lines were treated by BETd-260 at 100 nmol/L for 24 h. The protein levels of BRD2, BRD3 and BRD4 were examined by western blot analysis. Actin was used as a loading control. Data are representative of three independent experiments.

Article Snippet: The antibodies of BRD2 (A302-583A), BRD3 (A700-069), and BRD4 (A700-005) were purchased from Bethyl Laboratories (Shanghai, China).

Techniques: Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells

doi: 10.3389/fonc.2019.01471

Figure Lengend Snippet: BETd-260 induces BET degradation, triggers apoptosis, and inhibits proliferation in HCC xenograft tissue in mice. BALB/c mice bearing HepG2 and BEL-7402 xenograft tumors were treated by a single intravenous dose of 5 mg/kg BETd-260 (BETd) for 24 h or vehicle (Veh). (A) Two to three mice were sacrificed and tumor tissue was harvested. The expression of BRD2, BRD3, and BRD4, Mcl-1, Bad, activated caspase-3, cleaved PARP, and Ki 67 was examined by immunohistochemistry staining. Representative photographs were presented. (B) The percentages of HCC tumor cells positively stained with BRD2, BRD3, and BRD4, Mcl-1, Bad, cleaved caspase-3 (c-Casp3), cleaved PARP (c-PARP), and Ki 67 were quantified under microscopy, and plotted. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: The antibodies of BRD2 (A302-583A), BRD3 (A700-069), and BRD4 (A700-005) were purchased from Bethyl Laboratories (Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Staining, Microscopy

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