primary antibodies against brd4  (Bethyl)

Journal: Science Advances

Article Title: Tumor microenvironment–targeted PROTAC nanoparticle self-assembly broadly predicted by structural descriptors

doi: 10.1126/sciadv.adu2292

Figure Lengend Snippet: ( A ) Cell viability curves of NMC cells after treatment with free dBET6 or Fi-dBET6 NPs, as assessed by CellTiter-Glo. Dye-only control was treated at concentrations equivalent to that encapsulated into Fi-dBET6 NPs, as quantified by HPLC. N/A, not available. ( B ) Immunoblotting of BRD4 and β-actin in NMC cells after 24-hour treatment. ( C ) NanoBiT cells were treated with 0.01 μM of either free dBET6 or Fi-dBET6 NPs at t = 0. ( D ) NanoBiT cells were treated with free dBET6 or Fi-dBET6 NPs and washed three times with PBS, and the expression of BRD4 was monitored over 24 hours in the presence of Endurazine. Profiles are plotted as mean fractional relative luminescence units (RLU) values by normalizing to DMSO control. ( E ) Mean fluorescence intensity (MFI) of Fi-dBET6 NPs taken up by NMC cells following pretreatment with chlorpromazine (CPZ), a pharmacological inhibitor of endocytosis, as measured by flow cytometry. ( F ) Fi-dBET6 NP uptake and lysosomal colocalization 4 hours postwashout. ER, endoplasmic reticulum. Scale bars, 10 μm. The ROI (white box) is expanded in the second row of each condition. ( G ) Proposed mechanism of nanoPROTAC uptake and release. Data are means of technical replicates ± SEM [(A), (C), and (D)] where n = 3 or means of biological replicates ± SEM (E) where n = 3. Statistics were calculated using an ordinary one-way analysis of variance (ANOVA) with Dunnett’s post hoc test (E).

Article Snippet: Slides were then stained using a Discovery XT processor (Ventana Medical Systems-Roche) using primary antibodies against BRD4 (Bethyl, 50-156-1488), murine CD31 (Abcam, #ab182981), murine P-selectin (LSBio, #LSB3578) or human P-selectin (LSBio, #LSC78725).

Techniques: Control, Western Blot, Expressing, Fluorescence, Flow Cytometry

Journal: Science Advances

Article Title: Tumor microenvironment–targeted PROTAC nanoparticle self-assembly broadly predicted by structural descriptors

doi: 10.1126/sciadv.adu2292

Figure Lengend Snippet: ( A ) IF staining of P-selectin and CD31 in NMC tumor tissue. ( B ) Quantification of MFI (left) and representative fluorescence emission (right) of NP localization in tumors 24 hours postinjection of Fi-dBET6, Dex-dBET6 (untargeted control), free ICG, or free dBET6 (unlabeled). ( C ) Representative IHC of BRD4 in NMC tissue 48 hours posttreatment. ( D ) Quantification of BRD4 degradation in (C). ( E ) Pharmacokinetics of dBET6, as measured in plasma over time (data are means ± SEM where n = 4 biological replicates). ( F ) Nude mice engrafted subcutaneously with NMC cells were treated twice weekly with 15 mg/kg ip treatments of free dBET6, Fi-dBET6 NPs, vehicle, or untreated. ( G ) Tumor growth curves, ( H ) Kaplan-Meier survival curve, and ( I ) mouse weight change in NMC xenografts. Data are shown as individual biological replicates with means ± SEM, and statistics were calculated using one-way ANOVA with Dunnett’s post hoc test [(B) and (D)], multiple unpaired t tests with Holm-Sidak correction (E), unpaired t test of Fi-dBET6 versus free dBET6 (G), or Mantel-Cox survival analysis (H). ns, not significant; sc, subcutaneous.

Article Snippet: Slides were then stained using a Discovery XT processor (Ventana Medical Systems-Roche) using primary antibodies against BRD4 (Bethyl, 50-156-1488), murine CD31 (Abcam, #ab182981), murine P-selectin (LSBio, #LSB3578) or human P-selectin (LSBio, #LSC78725).

Techniques: Staining, Fluorescence, Control, Drug discovery, Clinical Proteomics

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Significantly recurrent breakpoints result in focal deletion of BRD4 regulatory regions (A) Top: breakpoint densities for PCAWG breast (BRCA), endometrial (EN) and ovarian (OV) cancers exhibiting rearrangements at the BRD4 locus, compared to an independent breast cancer dataset (Nik-Zainal et al. ); Center: somatic copy-number alterations (SCNAs) for pooled breast, ovarian, and endometrial cancer samples with BRD4 focal deletions. Bottom: alignment with H3K4me3 from breast (MCF-7) and ovarian (HMEC) cell lines. Genomic track: gene exons and intron as defined in RefSeq for hg19. (B and C) (B) Total copy number at CCNE1 locus (chromosome 19) between tumors with or without BRD4 focal deletions and (C) only breast, ovarian, and endometrial tumors; Wilcoxon rank-sum test. The box spans the interquartile range (IQR) with the median as a horizontal line and bars extending to 1.5 × IQR. (D) Proportion of tumors with or without amplifications spanning the BRD4 locus, comparing those with or without a concomitant BRD4 focal deletion.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques:

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Focal deletions of BRD4 significantly decrease BRD4 expression across isoforms (A) Volcano plot of global gene expression comparing BRD4 focal deletion tumors ( n = 22) with all other PCAWG tumors ( n = 2,636), Bonferroni-corrected cutoff of p < 0.1. Genes on chromosome 19 are displayed in bold type. (B) Expression of BRD4-L (ENST00000263377; BRD4-201) and BRD4-S(a) (ENST00000371835; BRD-203) isoforms in normal tissues as obtained from GTEx (TPM, transcripts per million). Two-tailed Wilcoxon signed rank test with Bonferroni-corrected p values as indicated. (C and D) Isoform-specific expression of BRD4 in PCAWG breast cancer (C) and ovarian (D) tumors, normalized to copy number, comparing BRD4-L (ENST00000263377; BRD4-201) with two BRD4-S isoforms (dominant BRD4-S(a): ENST00000371835, BRD-203, and secondary BRD4-S(b): ENST00000360016, BRD4-202) between tumors with or without BRD4 focal deletions (FPKM, fragments per kilobase of transcript per million fragments mapped). Wilcoxon rank-sum test. The box spans the IQR with the median as a horontal line and error bars extending to 1.5. (E) Coefficients of a linear model for BRD4 expression controlling for tumor type (non-breast, ovarian, or endometrial as baseline), absolute copy number, and focal deletion status (not deleted as baseline). Error bars represent the 95% confidence intervals for coefficient estimates from the linear model.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Expressing, Gene Expression, Two Tailed Test

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: BRD4 overexpression is toxic across multiple cancer types (A) Overview of an ORF screen, from which the effect of BRD4-L and BRD4-S(a) overexpression on cell proliferation can be assessed. (B) Log 2 -fold changes in cell proliferation for BRD4 constructs, expressed relative to the experimental early time point (ETP) of each screen. Negative changes reflect a detrimental effect on cell proliferation. ORF screens were conducted in breast (BRCA), ovarian (OV), neuroblastoma (NB), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), Ewing sarcoma (EWS), medulloblastoma (MB), and prostate adenocarcinoma (PRAD) cell lines. (C) ORF screen performance between BRD4-L and BRD4-S(a) isoforms for each cell line. Two-tailed t test. (D) Validation of BRD4 isoform expression in OVSAHO cells. Transcript expression is quantified relative to GFP control. Mean and standard deviation of three replicates; two-way ANOVA. (E) Immunoblot of BRD4-L and BRD4-S protein variants detected in OVSAHO cells transduced with BRD4 or control GFP overexpression constructs. Values represent levels of protein expression, normalized to glyceraldehyde 3-phosphate dehydrogenase expression and shown relative to parental cells (endogenous BRD4 levels). (F) Quantification of cell confluency in OVSAHO-Cas9 after transduction with BRD4-L, BRD4-S(a), or GFP control overexpressing constructs. Mean and standard deviation of three replicates, two-way ANOVA followed by Dunnett post-tests, controlling the Family-wise alpha threshold and confidence level. p values of the final time point are annotated.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Over Expression, Construct, Two Tailed Test, Biomarker Discovery, Expressing, Control, Standard Deviation, Western Blot, Transduction

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Use of CRISPR-Cas9 technology to mimic the effect of BRD4 focal deletions (A) Confirmation of Cas9 expression in OVSAHO cells by immunoblotting. (B) Fraction of GFP + cells detected in Cas9 activity assay. To measure Cas9 cutting efficiency, OVSAHO-Cas9 cells were transduced with a vector encoding GFP and sgGFP. After 10 days, a decreased population of GFP + cells was detected by flow cytometry, confirming Cas9 activity. (C) Intronic BRD4 regions targeted by CRISPR-Cas9 sgRNAs (inset not to scale due to length of intron 1). (D) CRISPR sequencing results for sgBRD4_region1 and sgBRD4_region2, showing the percentage of uncut, frameshift, and in-frame BRD4 reads when aligned to a reference sequence. (E) Expression of BRD4 isoforms following CRISPR-Cas9-mediated sgRNA cutting, relative to sgGFP control. Median and standard deviations of three replicates, two-way ANOVA. (F) Quantification of cell confluency in OVSAHO-Cas9 cells following CRISPR-Cas9-mediated sgRNA cutting of BRD4 regulatory regions or sgGFP control. Mean and standard deviation of three replicates, two-way ANOVA followed by Dunnett post-tests, controlling the Family-wise alpha threshold and confidence level. p values of the final time point are annotated. (G) Landscape of BRD4 dependency across ovarian cancers. Chronos scores (with mean and standard deviation) are shown for every ovarian cancer cell line included in the DepMap Public 23Q4 release. A score of −1 (median score for essential genes; red dashed line) is used as reference to indicate genetic dependency.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: CRISPR, Expressing, Western Blot, Activity Assay, Transduction, Plasmid Preparation, Flow Cytometry, Sequencing, Control, Standard Deviation

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet: Fine-tuned levels of BRD4 global expression are required for sustained cellular proliferation (A) Experimental workflow for mimicking concomitant focal BRD4 deletions occurring in BRD4 overexpressing ovarian cancer cells. (B) Rescue experiment by modulation of BRD4 expression. Quantification of cell confluency after CRISPR-Cas9-mediated gene ablation was measured in OVSAHO-Cas9 cells over-expressing BRD4-L (top) or BRD4-S(a) isoforms (bottom). Mean and standard error from three replicates, two-way ANOVA followed by Dunnett post-tests, controlling the Family-wise alpha threshold and confidence level. p values of the final time point are annotated. (C) Model of BRD4 expression regulation. In ovarian cancers, too-low or too-high expression levels are detrimental to cellular fitness. In BRD4 -amplified tumors, focal deletions serve as a mechanism to dampen global gene expression and rescue cellular proliferation.

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Expressing, CRISPR, Amplification, Gene Expression

Journal: Cell Genomics

Article Title: Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

doi: 10.1016/j.xgen.2025.100815

Figure Lengend Snippet:

Article Snippet: After blocking with 5% dry milk in TBS-T for 30 min, the membrane was incubated at 4°C overnight with primary antibodies against rabbit BRD4 (cat. no. 702448, ThermoFisher Scientific), rabbit BRD4-L (cat. no. A301-985A100, Bethyl Laboratories), mouse SpCas9 (cat. no. 14697, Cell Signaling Technology), or rabbit GAPDH (cat. No. 2118, Cell Signaling Technology).

Techniques: Virus, Expressing, Recombinant, Plasmid Preparation, Transfection, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software, CRISPR, Amplification, Sequencing

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 1. Illustration of ENCTAC performances under hypoxic condition for selective degradation of BRD4 proteins.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques:

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 2. Enzyme-derived click formation of heterobifunctional degraders of BRD4. (A) Chemical structure of GSH-responsive CRBN ligand (J266); GSH- and NTR- responsive CRBN ligand (JW4); cleaved J266; CBT-linked BRD4-targeting ligand (JQ1-CBT); click-induced BRD4 degrader (J252). (B) LC-MS spectra of NTR uncaging JW4 (10 μM) to form J266 at different time points in NTR (40 μg/ml) dissolved in phosphate-buffered saline (PBS; 10 mM) (pH 7.4). (C) Selectivity of JW4 toward a broad range of biological and chemical agents with or without NADH. (D) Time-dependent LC-MS spectra of click J252 formation under reducing reagent [tris(2-carbox- yethyl)phosphine (TCEP)] that cleave the J266 to induce cleaved J266 before the click reaction with JQ1-CBT in buffer solution. (E) Ratio of peak areas of click J252 and cleaved J266 to J266 over multiple time points in (D).

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Saline

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 3. Heterobifunctional degraders efficiently disrupt BRD4 protein. (A) Molecular dynamic simulation of J252 interacting with CRBN (gold) and BRD4 (blue) pro- teins. The position of the luciferin-based component shows no steric collision with BRD4 or CRBN. (snapshot at 1 ns). (B) Western blot analysis of BRD4 protein after treatment with J252 and ARV-825 in human embryonic kidney (HEK) 293T cells for 24 hours with indicated concentration and β-tubulin as internal controls.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Western Blot, Concentration Assay

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 4. Hypoxia-activated degradation of epigenetic BRD4 protein. (A) Illustration of intracellular hypoxic enzyme uncaging, formation of the degrader (click J252), and degradation of BRD4. (B) Western blot analysis of BRD4 protein levels after treatments with GSH-cleavable J266 (6 hours) and JQ1-CBT (12 hours) at indicated con- centration and different time points; GSH- and NTR-responsive JW4 (6 hours) with JQ1-CBT (12 hours) at the indicated concentration and for a different time period under hypoxia in HEK-293T cells. (C) Analysis of BRD4 protein levels under the combination of different control conditions for 12 hours (the inhibitor bortezomib incubated with cells for 2 hours before the addition of JW4 and JQ1-CBT). (D) Western blot analysis of BRD4 protein levels after treatments with J266 (6 hours) and JQ1-CBT (12 hours) or JW4 (6 hours) and JQ1-CBT (12 hours) in time- and concentration-dependent examinations of HeLa cancer cells; analysis of BRD4 protein levels after treatments with JQ1 (12 hours) in a concentration-dependent manner of hypoxia HeLa cells; and JW4 (6 hours) and JQ1-CBT (12 hours) in normoxia HeLa cells. (E) Extended concentration of degraders demonstrating the hook effect during incubation under hypoxia (representative results from two to three biological replications). β-Tubulin was used as in- ternal controls. (F) BRD4 degradation level over varied concentration as indicated in (E). Values represent triplicate means ± SD, normalized to nontreated cells, and baseline-corrected using immunoblots.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Western Blot, Concentration Assay, Control, Incubation

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 5. Hypoxic BRD4 degradation resulted in a change in the response of the cellular microenvironment and malfunction of cell growth. (A) Confocal imaging of HIF-1α immunostaining (green; λex = 488 nm and λem = 515/30 nm) after subjecting HeLa cells to the as-stated treatment. Nucleus was stained with Hoechst 33258 (blue; λex = 405 nm and λem = 460/50 nm), β-tubulin was stained with the fluorescent β-tubulin antibody (red; λex = 561 nm and λem = 617/20 nm). Scale bars, 40 μm. (B) Quantitative mean fluorescence intensity of HIF-1α after treatment and staining as indicated in (A). ka.u., kilo–arbitrary units. Values represent mean fluorescence intensity of three different cell areas ± SD. Western blot analysis of HIF-1α after treatment with different concentrations of JW4 and JQ1-CBT (C) or separated JW4, JQ1-CBT, JQ1, and J252 controls (12 hours) (D). (E) Western blot analysis of vascular endothelial growth factor (VEGF) and CA9 after hypoxia-activated ENCTAC treatment (12 hours). (F) Protein level of HIF-1α, VEGF, and CA9 after hypoxia ENCTAC (blue bar) and JQ1 inhibitor treatment (12 hours) (red bar). Values represent the average of duplicates and the range as error bars, normalized to nontreated cells, and baseline-corrected using immunoblots. (G) Immunoblot for c-Myc and glyceraldehyde-3-phosphate dehydro- genase (GAPDH) levels after hypoxia-activated ENCTAC treatment using JW4 and JQ1-CBT for 12 hours. (H) Immunoblot for poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) cleavage and GAPDH levels after similar treatment condition as in (G). (I) Confocal imaging of apoptosis cell death staining with annexin V (AnnV)/propodium iodide (PI) [AnnV (green), λex = 488 nm and λem = 520/30 nm; PI (red), λex = 561 nm and λem = 590/30 nm]. Hypoxic cells without ENCTAC treatment as control. BF, bright field. Scale bars, 40 μm. (J) Flow cytometry of apoptosis/necrosis-stained HeLa cells under treatments with JW4, JQ1-CBT individually, or in com- bination of JW4 and JQ1-CBT (10 μM) (12 hours). Quarter 1 (Q1) indicates the relative percentage of necrosis cells, Q2 indicates late apoptosis cells, Q3 indicates early apoptosis cells, and Q4 indicates live cells.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Imaging, Immunostaining, Staining, Fluorescence, Western Blot, Control, Flow Cytometry

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 6. In vivo degradation of BRD4 using ENCTACs to manipulate hypoxic zebrafish development. (A) Scheme of zebrafish early treatment with hypoxia-activated ENCTACs. (B) Western blot analysis of BRD4 level in hypoxia zebrafish after treatment with different concentrations of ENCTAC molecules. (C) Western blot analysis of HIF- 1α level in hypoxia zebrafish upon treatment with different concentrations of ENCTAC molecules or with inhibitor JQ1. (D) Bright-field images of zebrafish embryos under different conditions of indicated drug treatment at 36 hpf. Red arrowheads indicate yolk extension area. (E) Fluorescent imaging of zebrafish larvae phenotype with blood vessel trackers ( fli1:eGFPy1) in wild-type (WT), and von Hippel–Lindau (vhl) mutant (vhl−/−) larvae with and without ENCTAC treatment. Blood vessel tracker (green), λex = 488 nm and λem = 520/30 nm. Scale bar, 500 μm. (F) Fluorescent intensity spectra of blood vessel alignment along red line (I to IV) indicated in (E). (G) Statistical numbers of vascularization phenotypes in zebrafish larvae with and without ENCTAC treatments or with PROTAC, J252, or inhibitor JQ1 (10 μM).

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: In Vivo, Western Blot, Imaging, Mutagenesis

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 7. Tumor inhibition by ENCTACs assisted BRD4 degradation. (A) Schematic diagram shows the treatment of ENCTACs in the mouse melanoma xenograft model. (B) Fluorescence image of NTR activity in solid tumor. Near-infrared fluorescence reporter was intratumorally injected, and the image was taken 30 min later. (C) Immu- noblot for BRD4, c-Myc, HIF-1α, and GAPDH in tumor lysates collected from mice treated with JW4 and JQ1-CBT (5 mg/kg) mixture, JQ1 (5 mg/kg) alone, or vehicle twice for 4 hours. (D) Representative pictures and quantitative analysis of tumor volume (means ± SD) of vehicle-treated mice (n = 3) or mice treated with JW4, JQ1-CBT mixture (5 mg/kg; n = 4), or JQ1-treated mice (n = 3) for 5 days. (E) Relative tumor growth rate under different treatments as indicated for 5 days. (F) Immunohistochemistry staining with a vascular marker CD31 (green) and nucleus marker 4′,6-diamidino-2-phenylindole (DAPI) (blue) of tumors subjected to ENCTACs, (+)-JQ1, or vehicle control. (G) Quantification of total vascular area in tumors subjected to ENCTACs, (+)-JQ1, or vehicle control.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Inhibition, Fluorescence, Activity Assay, Injection, Immunohistochemistry, Staining, Marker, Control

Journal: Science advances

Article Title: Hypoxia deactivates epigenetic feedbacks via enzyme-derived clicking proteolysis-targeting chimeras.

doi: 10.1126/sciadv.abq2216

Figure Lengend Snippet: Fig. 8. Pharmacokinetic analysis and tumor inhibition studies of ENCTACs. (A and B) Plasma concentration–time profiles of JW4 (A) and JQ1-CBT (X = NH) (B) after intravenous dosing (5 mg/kg). (C) Schematic diagram for the treatment of ENCTACs in the xenograft mouse model with melanoma and HeLa tumor. (D) Representative images and quantitative analysis of tumor volume (means ± SD) of melanoma mice treated with vehicle (n = 4), JQ1 (5 mg/kg, n = 5), ARV-825 (5 mg/kg; n = 5), or JW4 + JQ1-CBT mixture (5 mg/kg; n = 5) for 6 days. (E) Western blot analysis of BRD4 levels in melanoma tumor after intravenous injection of vehicle, JQ1, ARV-825, or JW4 + JQ1-CBT (5 mg/kg). (F) Representative images and quantitative analysis of tumor volume (means ± SD) of HeLa tumor–bearing mice treated with vehicle control (n = 3), JQ1 (5 mg/kg, n = 3), ARV- 825 (5 mg/kg; n = 3), or JW4 + JQ1-CBT mixture (5 mg/kg; n = 3) for 8 days.

Article Snippet: Primary antibodies against BRD4 (#13440; 1:1000 dilution factor), HIF-1α (#36169; 1:1000 dilution factor), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #5174; 1:1000 dilution factor) were purchased from Cell Signaling Technologies; c-Myc (#700648; 1:1000 dilution factor), PARP1 (#MA5-15031; 1:500 dilution factor), VEGF (#P802; 1:500 dilution factor), and CA-IX (#MA5-29076; 1:1000 dilution factor) were purchased from Thermo Fisher Scientific; BRD4 (ab243862; 1:1000 dilution factor) and β-tubulin (ab6046; 1:10,000 dilution factor) were purchased from Abcam.

Techniques: Inhibition, Clinical Proteomics, Concentration Assay, Western Blot, Injection, Control

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Brd4 is required for peripheral T cells maintenance. (A, B) Flow cytometry showing the expression of CD3 (left) and quantification of total CD3 + T cells (right) in lymph node (A) and spleen (B) . (C, D) CD4 and CD8 expression determined by flow cytometry in lymph node (C) and spleen (D) (left), the absolute number and proportion of CD4 + T cells and CD8 + T cells in spleen and lymph node (right). (E, F) Naïve (CD44 - CD62L + ) and memory/activated (CD44 + ) T cells compartment analysis within CD4 + T (E) and CD8 + T cells (F) from freshly prepared splenocytes. Two-tailed unpaired t-test was used to analyze two independent groups. Results were indicated as mean ± sem (error bars). *p < 0.05; **p < 0.01; ***p < 0.001. NS, not significant. Each group includes at least 3 mice and each experiment repeats more than 3 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Flow Cytometry, Expressing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Brd4 is essential for naïve CD8 + T cells survival and homeostasis proliferation. (A) Flow cytometry showing the frequency (left) and quantification (right) of Annexin-V + CD8 + T cells. (B) Flow cytometry showing the frequency (left) and quantification of active-caspase3/7 + CD8 + T cells (right). (C) Purified Brd4 +/+ and Brd4 -/- naïve CD45.2 + CD8 + T cells were labeled with CellTrace Violet (CTV) and respectively transferred with naïve CD45.1 + CD8 + T cells (spike) into CD45.2 + recipient mice to monitor the survival capability of naïve CD8 + T cells for 7 days in vivo . Flow analysis of donor naïve CD8 + T cells in spleen and lymph node at day 7 post-transfer (left) and the relative ratio of recovered donor cells to spike (right). (D) Sorted naïve CD8 + T cells from Brd4 -/- and Brd4 +/+ mice were cultured with or without IL-7 for 3 days to test IL-7 triggered survival effect. (E) Experimental setup. Congenically marked Brd4 +/+ and Brd4 -/- naïve CD8 + T cells were labeled with CTV, mixed at a 1:1 ratio, and then transferred into Rag1 mice to monitor cell proliferation at day 7 post-transfer. (F) Relative frequency of donor cells at transfer. (G) Representative flow analysis of donor cells recovered at day 7 post-transfer. (H) The quantification of donor CD8 + T cells at day 7 post transfer. Two-tailed unpaired t -test was used to analyze two independent groups, while paired Student’s t -test was used when sample being compared from same mouse. Results were indicated as mean ± sem (error bars). **p < 0.01; ***p < 0.001. NS, not significant. Each group includes at least 3 mice and each experiment repeats more than 3 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Flow Cytometry, Purification, Labeling, In Vivo, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Brd4 promotes the clonal expansion of CD8 + T cells during viral infection. (A) Experiment setup. (B) Brd4 +/+ and Brd4 -/- effector P14 + CD8 + T cells recovered at day 8 in indicated tissues (left) and the quantification of effector P14 + CD8 + T cells post-infection (right). (C) Brd4 +/+ and Brd4 -/- memory P14 + CD8 + T cells recovered at day 35 in indicated tissues (left) and the quantification of memory P14 + CD8 + T cells post-infection (right). Paired Student’s t -test was used to analyze data. Results were indicated as mean ± sem (error bars). *p < 0.05; **p < 0.01; ***p < 0.001. Each group includes at least 4 mice and each experiment repeats more than 3 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Infection

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Brd4 regulates migration- and glucose metabolism-related gene transcription. (A) GSEA enrichment analysis of genes involved in glucose metabolism and cell migration. (B) Heat map showing the downregulated genes in CD8 + T cells after Brd4 deletion and RT-qPCR confirmation of downregulated genes. (C) Flow cytometry showing the expression of CD62L in naïve CD8 + T cells. (D) Flow cytometry showing the expression of LFA-1 in naïve CD8 + T cells. (E) Flow cytometry analysis of GLUT1 expression in naïve CD8 + T cells. (F) CUT&Tag analysis showing the binding sites of BRD4 in Slc2a1 gene loci (left) and ChIP-qPCR confirmation of the enrichment of BRD4 in Slc2a1 loci in CD8 + T cells (right). (G) Intracellular staining for Myc detection in Brd4 +/+ and Brd4 -/- naïve and activated CD8 + T cells. (H) ChIP-qPCR confirmation of the enrichment of BRD4 in Myc locus in CD8 + T cells. The data was analyzed by two-tailed unpaired t -test between two independent groups. Results were indicated as mean ± sem (error bars). **p < 0.01; ***p < 0.001; ****p < 0.0001. Each experiment repeats more than 3 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Migration, Quantitative RT-PCR, Flow Cytometry, Expressing, Binding Assay, ChIP-qPCR, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Brd4 ensures CD8 + T cells homing and mitochondrial glucose oxidation. (A) Experiment setup. Purified Brd4 +/+ (CD45.1 - CD45.2 + ) and Brd4 -/- (CD45.1 + CD45.2 + ) naïve CD8 + T cells were mixed at a 1:1 ratio and then transferred into CD45.1 + CD45.2 - recipient mice. Donor cell recovery was checked in indicated tissues to assess their homing ability after 18h post-transfer. (B) Relative recovery of donor naïve CD8 + T cells in all checked organs. (C) RT-qPCR analysis of Slc2a1 transcription at distinct time point after CD8 + T cell receiving TCR stimulation. (D) Glucose uptake measurement in naïve and in vitro -activated CD8 + T cells from Brd4 +/+ and Brd4 -/- mice. (E) Oxidative phosphorylation in Brd4 +/+ and Brd4 -/- naïve CD8 + T cells was determined by measuring oxygen consuming rate after adding indicated compounds. (F) Schematic of in vitro migration assay in transwell plate (left) and the migration efficiency of Brd4 +/+ and Brd4 -/- naïve CD8 + T cells in response to CCL21 chemotaxis (right). Two-tailed unpaired t -test was used to analyze two independent groups, while paired Student’s t -test was used when sample being compared from same mouse. To compare multiple groups, we used an analysis of variance (ANOVA). Results were indicated as mean ± sem (error bars). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. NS, not significant. Each group includes at least 3 mice and each experiment repeats more than 2 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Purification, Cell Recovery, Quantitative RT-PCR, In Vitro, Phospho-proteomics, Migration, Chemotaxis Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Brd4 promotes CD8 + T cell proliferation through inducing glycolysis. (A) Purified Brd4 +/+ and Brd4 -/- naïve CD8 + T cells were activated with anti-CD3/28 antibody for 24 hours to assess cell activation. Flow cytometry showing the expression of CD71 after stimulation (left) and the quantification of CD71 MFI (right). (B) Flow analysis of cell size of in vitro -activated CD8 + T cells. (C) Purified Brd4 +/+ and Brd4 -/- naïve CD8 + T cells were labeled with CellTrace Violet (CTV) and activated with anti-CD3/28 antibody in the presence or absence of IL-2 for 3 days to assess cell proliferation. Flow cytometry showing CTV dilution. (D) Intracellular staining showing the expression of Ki-67 in Brd4 +/+ and Brd4 -/- CD8 + T cells after activation for 24h (left) and the quantification of Ki-67 + CD8 + T cells (right). (E) qPCR analysis showing the differentially expressed glycolytic genes in Brd4 +/+ and Brd4 -/- CD8 + T cells. (F) Chart showing glycolysis measurement of in vitro -activated CD8 + T cells (left) and the quantification of the basal and maximal glycolysis (right). Two-tailed unpaired t -test was used to analyze two independent groups. Results were indicated as mean ± sem (error bars). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Each group includes at least 3 mice and each experiment repeats more than 2 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Purification, Activation Assay, Flow Cytometry, Expressing, In Vitro, Labeling, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Targeting Brd4 with inhibitors dampens effector CD8 + T cells-mediated antiviral immunity. (A) Experiment design. (B) Flow cytometry showing the expression of CD8 (left), the quantification of total CD8 + T cells (middle) and the quantification of LCMVgp33 epitope-specific CD8 + T cell numbers (right). (C) Flow cytometry showing the expression of KLRG1 and CD127 in LCMVgp33 epitope-specific CD8 + T cells from mice treated with JQ1 or DMSO at day 8 post-infection (left) and the frequency and number of each subset (right). (D, E) Flow cytometry analysis of indicated cytokines production after peptide stimulation in CD8 + T cells from mice treated with JQ1 or DMSO (left) and the number and frequency (right) of cytokine-producing CD8 + T cells. The data were analyzed by two-tailed unpaired t -test between two independent groups. Results were indicated as mean ± sem (error bars). *p < 0.05; **p < 0.01; ***p < 0.001. Each group includes at least 4 mice and each experiment repeats more than 2 times.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Flow Cytometry, Expressing, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Brd4 Regulates the Homeostasis of CD8 + T-Lymphocytes and Their Proliferation in Response to Antigen Stimulation

doi: 10.3389/fimmu.2021.728082

Figure Lengend Snippet: Schematics showing that Brd4 regulates CD8 + T cell homeostasis and proliferation through controlling glucose metabolism. Brd4 maintains naïve CD8 + T cells homeostasis by regulating the survival and trafficking of naïve CD8 + T cells in steady-state. Brd4 promotes efficient glucose uptake by upregulating GLUT1 (glucose transporter 1) and Myc expression, which glucose oxidation in mitochondria fuels naïve CD8 + T cells survival and trafficking. Upon activation, activated CD8 + T cells increase glucose uptake to meet the bioenergetic and biosynthetic demands, which includes quick proliferation, survival and effector differentiation.

Article Snippet: First, CD8 + T cell was incubated with primary antibody against Brd4 (1:50; Active motif AB_2615059) and then with guinea pig anti-rabbit secondary antibody (1:100; Antibodies Online ABIN101961).

Techniques: Expressing, Activation Assay