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antibodies against brd4 (Bethyl)

Name: antibodies against brd4
Brand: Bethyl
Rating: 95 (48 reviews)

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Bethyl antibodies against brd4

<t>E2F1-BRD4</t> binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human <t>BRD4</t> BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Antibodies Against Brd4, supplied by Bethyl, used in various techniques. Bioz Stars score: 95/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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antibodies against brd4 - by Bioz Stars, 2026-02

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1) Product Images from "E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells"

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkaf1513

E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human BRD4 BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Figure Legend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human BRD4 BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Techniques Used: Binding Assay, In Vitro, Staining, SDS-Gel, Purification, Modification, Methylation, Synthesized, Sequencing, Incubation

E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in cells. ( A ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) were transfected into DU145 SETD6 WT and KO cells. The GFP-tagged BRD4 was purified by GFP-trap and analyzed by western-blot with an anti-GFP antibody. Co-purification of Flag-E2F1 was determined by anti-Flag antibody. Equal loading of cell lysate isolated from transfected DU145 SETD6 WT or KO was verified by western-blot analysis against β-actin, GFP, and Flag. ( B ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) WT or K117R were transfected into in DU145 SETD6 KO cells. Some of the transfected cells were treated with JQ1-Bromodomain-Kac binding inhibitor (5 µM) or DMSO as control. GPF-trap and western-blot analysis was conducted as in panel A. (C–E) Interaction of BRD4 and E2F1 investigated by PLA. All experiments were conducted in DU145 cells. Exemplary microscopy images are shown. Scale bar: 10 µm. PLA signal quantification (PLA dots per nucleus, AU) for each sample is shown on the right. Statistical analysis was performed using Student’s t -test in GraphPad (**** P < .0001). ( C ) Interaction of endogenous BRD4 and Flag-E2F1 in the absence and the presence of the SAHA deacetylase inhibitor (20 µM) for 5 h (Flag-E2F1). Negative control (Neg) refers to reaction conducted without addition of Flag primary antibody. The interaction of BRD4 and E2F1 was detected and it was shown to be stimulated by increasing acetylation levels after SAHA treatment. Number of analyzed cells: 183, 132, 223. ( D ) Detection of the interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells (Control) and SETD6 KO cells (KO1 and KO2). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed cells: 67, 236, 104, 91. ( E ) Interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells and with overexpression of GFP (GFP empty) or GFP-SETD6 (GFP-SETD6). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed GFP positive cells: 19, 18, 28.

Figure Legend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in cells. ( A ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) were transfected into DU145 SETD6 WT and KO cells. The GFP-tagged BRD4 was purified by GFP-trap and analyzed by western-blot with an anti-GFP antibody. Co-purification of Flag-E2F1 was determined by anti-Flag antibody. Equal loading of cell lysate isolated from transfected DU145 SETD6 WT or KO was verified by western-blot analysis against β-actin, GFP, and Flag. ( B ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) WT or K117R were transfected into in DU145 SETD6 KO cells. Some of the transfected cells were treated with JQ1-Bromodomain-Kac binding inhibitor (5 µM) or DMSO as control. GPF-trap and western-blot analysis was conducted as in panel A. (C–E) Interaction of BRD4 and E2F1 investigated by PLA. All experiments were conducted in DU145 cells. Exemplary microscopy images are shown. Scale bar: 10 µm. PLA signal quantification (PLA dots per nucleus, AU) for each sample is shown on the right. Statistical analysis was performed using Student’s t -test in GraphPad (**** P < .0001). ( C ) Interaction of endogenous BRD4 and Flag-E2F1 in the absence and the presence of the SAHA deacetylase inhibitor (20 µM) for 5 h (Flag-E2F1). Negative control (Neg) refers to reaction conducted without addition of Flag primary antibody. The interaction of BRD4 and E2F1 was detected and it was shown to be stimulated by increasing acetylation levels after SAHA treatment. Number of analyzed cells: 183, 132, 223. ( D ) Detection of the interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells (Control) and SETD6 KO cells (KO1 and KO2). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed cells: 67, 236, 104, 91. ( E ) Interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells and with overexpression of GFP (GFP empty) or GFP-SETD6 (GFP-SETD6). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed GFP positive cells: 19, 18, 28.

Techniques Used: Binding Assay, Transfection, Purification, Western Blot, Copurification, Isolation, Control, Microscopy, Histone Deacetylase Assay, Negative Control, Over Expression

Figure Legend Snippet: E2F1-BRD4 co-occurence is observed in SETD6 KO but not SETD6 WT cells. ( A ) Heatmap of RPKM-normalized E2F1 ChIP-seq signals at E2F1 peaks (±0.8 kb) showing differential chromatin binding of E2F1 in SETD6 WT and KO cells stably expressing Flag-E2F1. The third heatmap shows BRD4 Chip-seq signals in a prostate cancer cell line (SRR1170714) using the same clustering. See also . ( B ) Example browser views showing ChIP-seq of BRD4 (SRR1170714, green) and E2F1 in SETD6 WT and KO cells. See also for additional examples. ( C ) Correlation analysis of E2F1 binding in SETD6 WT and KO cells with the literature BRD4 chromatin binding profile used in panel (A). E2F1 and BRD4 signals were determined in the E2F1 peak regions shown in panel (A) and their correlation was determined. ( D ) Bar-graph showing the slope of the correlation line of BRD4 and E2F1 binding signals in SETD6 WT or KO cells determined using three BRD4 ChIP-seq data sets (datasets SRR1170714, SRR5467129, and SRR5467130). The corresponding analyses are shown in panel (C) and . P -value determined by two-flanked t -test assuming equal variance.

Techniques Used: ChIP-sequencing, Binding Assay, Stable Transfection, Expressing

Promoter and enhancer binding of BRD4 at genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. ( A ) Average of aggregated BRD4 signals at promoter and enhancer elements of genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. Note stronger binding in SETD6 KO cells. P -value determined by two-flanked t -test assuming equal variance. ( B ) Representative genome browser views showing co-occupancy of BRD4 and E2F1 at five genomic regions in SETD6 KO cells: C6orf226, TBCC, RPL21, RPL38, and MYC. ChIP-seq tracks were visualized using IGV (version 2.13.1) displaying BRD4 (SRR1170714, green), E2F1 in SETD6 WT (blue), and E2F1 in SETD6 KO (red) DU145 cells. ( C ) ChIP from SETD6 WT and KO DU145 cells performed using a BRD4-specific antibody to enrich BRD4-bound chromatin fragments. IgG was used as a negative control to assess the specificity of the immunoprecipitation. BRD4 occupancy was evaluated by qPCR at the same loci as shown in panel (B). Two independent biological replicates with three technical repeats were performed. Statistical significance was determined using a two-tailed t -test assuming equal variance. The negative controls RPL21 and RPL28 did not yield a detectable signal. Note the elevated BRD4 binding in SETD6 KO context. ( D ) RT-qPCR analysis of the relative expression of the five target genes shown in panel (B) in untreated SETD6 WT and KO DU145 cells (control) as well as after addition of DMSO and JQ1. Note the strong effect of JQ1 on gene expression in SETD6 KO cells.

Figure Legend Snippet: Promoter and enhancer binding of BRD4 at genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. ( A ) Average of aggregated BRD4 signals at promoter and enhancer elements of genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. Note stronger binding in SETD6 KO cells. P -value determined by two-flanked t -test assuming equal variance. ( B ) Representative genome browser views showing co-occupancy of BRD4 and E2F1 at five genomic regions in SETD6 KO cells: C6orf226, TBCC, RPL21, RPL38, and MYC. ChIP-seq tracks were visualized using IGV (version 2.13.1) displaying BRD4 (SRR1170714, green), E2F1 in SETD6 WT (blue), and E2F1 in SETD6 KO (red) DU145 cells. ( C ) ChIP from SETD6 WT and KO DU145 cells performed using a BRD4-specific antibody to enrich BRD4-bound chromatin fragments. IgG was used as a negative control to assess the specificity of the immunoprecipitation. BRD4 occupancy was evaluated by qPCR at the same loci as shown in panel (B). Two independent biological replicates with three technical repeats were performed. Statistical significance was determined using a two-tailed t -test assuming equal variance. The negative controls RPL21 and RPL28 did not yield a detectable signal. Note the elevated BRD4 binding in SETD6 KO context. ( D ) RT-qPCR analysis of the relative expression of the five target genes shown in panel (B) in untreated SETD6 WT and KO DU145 cells (control) as well as after addition of DMSO and JQ1. Note the strong effect of JQ1 on gene expression in SETD6 KO cells.

Techniques Used: Binding Assay, ChIP-sequencing, Negative Control, Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Expressing, Control, Gene Expression

Summary of the results of this study. SETD6 monomethylates E2F1 at K117. This methylation disrupts the E2F1–BRD4 interaction leading to different target loci being bound by both factors. In the absence of K117 methylation, E2F1 is acetylated at K117 and K120 leading to BRD4 binding and a concerted engagement of both protein at genomic target sites. As a consequence, methylated and unmethylated E2F1 regulates distinct gene sets in prostate cancer cells.

Figure Legend Snippet: Summary of the results of this study. SETD6 monomethylates E2F1 at K117. This methylation disrupts the E2F1–BRD4 interaction leading to different target loci being bound by both factors. In the absence of K117 methylation, E2F1 is acetylated at K117 and K120 leading to BRD4 binding and a concerted engagement of both protein at genomic target sites. As a consequence, methylated and unmethylated E2F1 regulates distinct gene sets in prostate cancer cells.

Techniques Used: Methylation, Binding Assay

Image Search Results

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in vitro . ( A ) Crystal structure of the human BRD4 BD1 (sandy-brown) in complex with an acetylated K117ac/K120ac E2F1 peptide (blue) (PDB 6ULS) showing the key interaction of K117ac with BD1. ( B ) Coomassie BB stained 12% SDS gel of the purified GST tagged truncated BRD4 (2-477 aa) protein including BD1 and BD2 (BD1/2) as well as the purified BD1 domain (2-220 aa). The GST-tagged BRD4 proteins are marked with asterisks. ( C ) Binding of the GST-BRD4 BD1/2 to modified E2F1 peptides. 15 aa long E2F1 peptides with different combinations of unmodified, acetylated, and methylated K117 and K120 were synthesized on peptide SPOT arrays. The sequence of each peptide is listed in the table. Peptide arrays were incubated with 5 nM GST-BRD4 BD1/2 and binding was detected using a GST-specific antibody. The bar diagram shows the binding of E2F1-BRD4 to K117ac/K120ac and K117me/K120ac observed in three independent experiments. The bars represent the averages. The P- value was determined by two flanked t ‐test with equal variance. ( D ) Same as in panel (C), but GST-BRD4 BD1 was used. Additional data are provdied in .

Article Snippet: PLA Duolink assays were performed according to the manufacturer’s instructions (Sigma) using antibodies against BRD4 (Bethyl, A700-004), E2F1 (SantaCruz, SC-251) and Flag (Sigma, F1804) overnight at 4°C.

Techniques: Binding Assay, In Vitro, Staining, SDS-Gel, Purification, Modification, Methylation, Synthesized, Sequencing, Incubation

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 binding is lost with K117 monomethylated E2F1 in cells. ( A ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) were transfected into DU145 SETD6 WT and KO cells. The GFP-tagged BRD4 was purified by GFP-trap and analyzed by western-blot with an anti-GFP antibody. Co-purification of Flag-E2F1 was determined by anti-Flag antibody. Equal loading of cell lysate isolated from transfected DU145 SETD6 WT or KO was verified by western-blot analysis against β-actin, GFP, and Flag. ( B ) GFP-tagged BRD4 (2-477) and Flag-E2F1 (2-437) WT or K117R were transfected into in DU145 SETD6 KO cells. Some of the transfected cells were treated with JQ1-Bromodomain-Kac binding inhibitor (5 µM) or DMSO as control. GPF-trap and western-blot analysis was conducted as in panel A. (C–E) Interaction of BRD4 and E2F1 investigated by PLA. All experiments were conducted in DU145 cells. Exemplary microscopy images are shown. Scale bar: 10 µm. PLA signal quantification (PLA dots per nucleus, AU) for each sample is shown on the right. Statistical analysis was performed using Student’s t -test in GraphPad (**** P < .0001). ( C ) Interaction of endogenous BRD4 and Flag-E2F1 in the absence and the presence of the SAHA deacetylase inhibitor (20 µM) for 5 h (Flag-E2F1). Negative control (Neg) refers to reaction conducted without addition of Flag primary antibody. The interaction of BRD4 and E2F1 was detected and it was shown to be stimulated by increasing acetylation levels after SAHA treatment. Number of analyzed cells: 183, 132, 223. ( D ) Detection of the interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells (Control) and SETD6 KO cells (KO1 and KO2). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed cells: 67, 236, 104, 91. ( E ) Interaction of endogenous BRD4 and endogenous E2F1 in the presence of 40 µM SAHA for 5 h in DU145 cells and with overexpression of GFP (GFP empty) or GFP-SETD6 (GFP-SETD6). Negative control (Negative) refers to reaction conducted without addition of E2F1 primary antibody. Number of analyzed GFP positive cells: 19, 18, 28.

Techniques: Binding Assay, Transfection, Purification, Western Blot, Copurification, Isolation, Control, Microscopy, Histone Deacetylase Assay, Negative Control, Over Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: E2F1-BRD4 co-occurence is observed in SETD6 KO but not SETD6 WT cells. ( A ) Heatmap of RPKM-normalized E2F1 ChIP-seq signals at E2F1 peaks (±0.8 kb) showing differential chromatin binding of E2F1 in SETD6 WT and KO cells stably expressing Flag-E2F1. The third heatmap shows BRD4 Chip-seq signals in a prostate cancer cell line (SRR1170714) using the same clustering. See also . ( B ) Example browser views showing ChIP-seq of BRD4 (SRR1170714, green) and E2F1 in SETD6 WT and KO cells. See also for additional examples. ( C ) Correlation analysis of E2F1 binding in SETD6 WT and KO cells with the literature BRD4 chromatin binding profile used in panel (A). E2F1 and BRD4 signals were determined in the E2F1 peak regions shown in panel (A) and their correlation was determined. ( D ) Bar-graph showing the slope of the correlation line of BRD4 and E2F1 binding signals in SETD6 WT or KO cells determined using three BRD4 ChIP-seq data sets (datasets SRR1170714, SRR5467129, and SRR5467130). The corresponding analyses are shown in panel (C) and . P -value determined by two-flanked t -test assuming equal variance.

Techniques: ChIP-sequencing, Binding Assay, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Promoter and enhancer binding of BRD4 at genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. ( A ) Average of aggregated BRD4 signals at promoter and enhancer elements of genes preferentially bound by E2F1 and upregulated in SETD6 WT or KO context. Note stronger binding in SETD6 KO cells. P -value determined by two-flanked t -test assuming equal variance. ( B ) Representative genome browser views showing co-occupancy of BRD4 and E2F1 at five genomic regions in SETD6 KO cells: C6orf226, TBCC, RPL21, RPL38, and MYC. ChIP-seq tracks were visualized using IGV (version 2.13.1) displaying BRD4 (SRR1170714, green), E2F1 in SETD6 WT (blue), and E2F1 in SETD6 KO (red) DU145 cells. ( C ) ChIP from SETD6 WT and KO DU145 cells performed using a BRD4-specific antibody to enrich BRD4-bound chromatin fragments. IgG was used as a negative control to assess the specificity of the immunoprecipitation. BRD4 occupancy was evaluated by qPCR at the same loci as shown in panel (B). Two independent biological replicates with three technical repeats were performed. Statistical significance was determined using a two-tailed t -test assuming equal variance. The negative controls RPL21 and RPL28 did not yield a detectable signal. Note the elevated BRD4 binding in SETD6 KO context. ( D ) RT-qPCR analysis of the relative expression of the five target genes shown in panel (B) in untreated SETD6 WT and KO DU145 cells (control) as well as after addition of DMSO and JQ1. Note the strong effect of JQ1 on gene expression in SETD6 KO cells.

Techniques: Binding Assay, ChIP-sequencing, Negative Control, Immunoprecipitation, Two Tailed Test, Quantitative RT-PCR, Expressing, Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells

doi: 10.1093/nar/gkaf1513

Figure Lengend Snippet: Summary of the results of this study. SETD6 monomethylates E2F1 at K117. This methylation disrupts the E2F1–BRD4 interaction leading to different target loci being bound by both factors. In the absence of K117 methylation, E2F1 is acetylated at K117 and K120 leading to BRD4 binding and a concerted engagement of both protein at genomic target sites. As a consequence, methylated and unmethylated E2F1 regulates distinct gene sets in prostate cancer cells.

Techniques: Methylation, Binding Assay

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

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

( 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.

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.

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

Analysis of correlations with each other among CENP-F and related genes. (A) The PPI network for CENP-F, CDK1, CDK2, CDK7 and BRD4 in human species. (B) The PPI network for CENP-F, CDK1, CDK2, CDK7 and BRD4 in mouse species. (C) The correlation between CENP-F and CDK1 in HCC. (D) The correlation between BRD4 and CDK1 in HCC. (E) The correlation between CENP-F and CDK2 in HCC. (F) The correlation between BRD4 and CDK2 in HCC. (G) The correlation between CENP-F and BRD4 in HCC

Journal: Discover Oncology

Article Title: CENP-F promotes HCC cell proliferation mediated by super enhancer reader BRD4

doi: 10.1007/s12672-025-03785-5

Figure Lengend Snippet: Analysis of correlations with each other among CENP-F and related genes. (A) The PPI network for CENP-F, CDK1, CDK2, CDK7 and BRD4 in human species. (B) The PPI network for CENP-F, CDK1, CDK2, CDK7 and BRD4 in mouse species. (C) The correlation between CENP-F and CDK1 in HCC. (D) The correlation between BRD4 and CDK1 in HCC. (E) The correlation between CENP-F and CDK2 in HCC. (F) The correlation between BRD4 and CDK2 in HCC. (G) The correlation between CENP-F and BRD4 in HCC

Article Snippet: Antibodies against BRD4 and c-Myc were purchased from Affinity Biosciences (DF2905, Liyang, China) and Proteintech (67447-1-Ig, Wuhan, China), respectively.

Techniques:

The mRNA expression of CENP-F and related genes in HCC. (A) The mRNA expression levels of CENP-F in HCC. (B) The mRNA expression levels of CDK1 in HCC. (C) The mRNA expression levels of CDK2 in HCC. (D) The mRNA expression levels of BRD4 in HCC. *<0.05, **<0.01, *** <0.001, ****<0.0001 vs. match control. Statistical significance was tested by Student’s t -test. Data represent mean ± SEM of more than three independent experiments

Journal: Discover Oncology

Article Title: CENP-F promotes HCC cell proliferation mediated by super enhancer reader BRD4

doi: 10.1007/s12672-025-03785-5

Figure Lengend Snippet: The mRNA expression of CENP-F and related genes in HCC. (A) The mRNA expression levels of CENP-F in HCC. (B) The mRNA expression levels of CDK1 in HCC. (C) The mRNA expression levels of CDK2 in HCC. (D) The mRNA expression levels of BRD4 in HCC. *<0.05, **<0.01, *** <0.001, ****<0.0001 vs. match control. Statistical significance was tested by Student’s t -test. Data represent mean ± SEM of more than three independent experiments

Article Snippet: Antibodies against BRD4 and c-Myc were purchased from Affinity Biosciences (DF2905, Liyang, China) and Proteintech (67447-1-Ig, Wuhan, China), respectively.

Techniques: Expressing, Control

The expression of mRNA and protein levels of related genes after knockdown CENP-F and overexpression of CENP-F in vitro. (A) RT-qPCR analysis of CENP-F expression in HepG2 cell line among the normal contral (NC) group, shCENP-F group, empty vector group and overexpression group. (B) RT-qPCR analysis of CDK1 expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (C) RT-qPCR analysis of CDK2 expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (D) RT-qPCR analysis of BRD4 expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (E) RT-qPCR analysis of c-Myc expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (F) RT-qPCR analysis of CENP-F expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (G) RT-qPCR analysis of CDK1 expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (H) RT-qPCR analysis of CDK2 expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (I) RT-qPCR analysis of BRD4 expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (J) RT-qPCR analysis of c-Myc expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. K. Western blotting analysis of CENP-F, CDK1, CDK2, BRD4 and c-Myc expression in HepG2 cell line. L. Western blotting analysis of CENP-F, CDK1, CDK2, BRD4 and c-Myc expression in Hep3B cell line. *<0.05, **<0.01, *** <0.001, ****<0.0001 vs. match control. Statistical significance was tested by Student’s t -test. Data represent mean ± SEM of three independent experiments

Journal: Discover Oncology

Article Title: CENP-F promotes HCC cell proliferation mediated by super enhancer reader BRD4

doi: 10.1007/s12672-025-03785-5

Figure Lengend Snippet: The expression of mRNA and protein levels of related genes after knockdown CENP-F and overexpression of CENP-F in vitro. (A) RT-qPCR analysis of CENP-F expression in HepG2 cell line among the normal contral (NC) group, shCENP-F group, empty vector group and overexpression group. (B) RT-qPCR analysis of CDK1 expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (C) RT-qPCR analysis of CDK2 expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (D) RT-qPCR analysis of BRD4 expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (E) RT-qPCR analysis of c-Myc expression in HepG2 cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (F) RT-qPCR analysis of CENP-F expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (G) RT-qPCR analysis of CDK1 expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (H) RT-qPCR analysis of CDK2 expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (I) RT-qPCR analysis of BRD4 expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. (J) RT-qPCR analysis of c-Myc expression in Hep3B cell line among the NC group, shCENP-F group, empty vector group and overexpression group. K. Western blotting analysis of CENP-F, CDK1, CDK2, BRD4 and c-Myc expression in HepG2 cell line. L. Western blotting analysis of CENP-F, CDK1, CDK2, BRD4 and c-Myc expression in Hep3B cell line. *<0.05, **<0.01, *** <0.001, ****<0.0001 vs. match control. Statistical significance was tested by Student’s t -test. Data represent mean ± SEM of three independent experiments

Article Snippet: Antibodies against BRD4 and c-Myc were purchased from Affinity Biosciences (DF2905, Liyang, China) and Proteintech (67447-1-Ig, Wuhan, China), respectively.

Techniques: Expressing, Knockdown, Over Expression, In Vitro, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Control

Knockdown CENP-F and BRD4 inhibited HCC cell proliferation in vivo. (A) Subcutaneous tumor model of HCC among NC group, shBRD4 and shCENP-F group. (B) Subcutaneous tumor size of HCC among NC group, shBRD4 and shCENP-F group. (C) Tumor growth volume comparison among NC group, shBRD4 and shCENP-F group. (D) Live imaging of tumor tissue after CENP-F inhibition. (E) Live imaging of tumor tissue after BRD4 inhibition. (F) RT-qPCR analysis of CENP-F expression in tumor tissue between the NC group and the shCENP-F group. (G) RT-qPCR analysis of CDK1 expression in tumor tissue between the NC group and the shCENP-F group. (H) RT-qPCR analysis of CDK2 expression in tumor tissue between the NC group and the shCENP-F group. (I) RT-qPCR analysis of BRD4 expression in tumor tissue between the NC group and the shCENP-F group. (J) RT-qPCR analysis of c-Myc expression in tumor tissue between the NC group and the shCENP-F group. K. RT-qPCR analysis of BRD4 expression in tumor tissue between the NC group and the shBRD4 group. L. RT-qPCR analysis of c-Myc expression in tumor tissue between the NC group and the shBRD4 group. M. Western blotting analysis of CENP-F, CDK1, CDK2, BRD4 and c-Myc expression in tumor tissue between the NC group and the shCENP-F group. N. Western blotting analysis of BRD4 and c-Myc expression in tumor tissue between the NC group and the shBRD4 group. *<0.05, **<0.01, *** <0.001, ****<0.0001 vs. match control. Statistical significance was tested by Student’s t -test. Data represent mean ± SEM of three or six independent experiments

Journal: Discover Oncology

Article Title: CENP-F promotes HCC cell proliferation mediated by super enhancer reader BRD4

doi: 10.1007/s12672-025-03785-5

Figure Lengend Snippet: Knockdown CENP-F and BRD4 inhibited HCC cell proliferation in vivo. (A) Subcutaneous tumor model of HCC among NC group, shBRD4 and shCENP-F group. (B) Subcutaneous tumor size of HCC among NC group, shBRD4 and shCENP-F group. (C) Tumor growth volume comparison among NC group, shBRD4 and shCENP-F group. (D) Live imaging of tumor tissue after CENP-F inhibition. (E) Live imaging of tumor tissue after BRD4 inhibition. (F) RT-qPCR analysis of CENP-F expression in tumor tissue between the NC group and the shCENP-F group. (G) RT-qPCR analysis of CDK1 expression in tumor tissue between the NC group and the shCENP-F group. (H) RT-qPCR analysis of CDK2 expression in tumor tissue between the NC group and the shCENP-F group. (I) RT-qPCR analysis of BRD4 expression in tumor tissue between the NC group and the shCENP-F group. (J) RT-qPCR analysis of c-Myc expression in tumor tissue between the NC group and the shCENP-F group. K. RT-qPCR analysis of BRD4 expression in tumor tissue between the NC group and the shBRD4 group. L. RT-qPCR analysis of c-Myc expression in tumor tissue between the NC group and the shBRD4 group. M. Western blotting analysis of CENP-F, CDK1, CDK2, BRD4 and c-Myc expression in tumor tissue between the NC group and the shCENP-F group. N. Western blotting analysis of BRD4 and c-Myc expression in tumor tissue between the NC group and the shBRD4 group. *<0.05, **<0.01, *** <0.001, ****<0.0001 vs. match control. Statistical significance was tested by Student’s t -test. Data represent mean ± SEM of three or six independent experiments

Article Snippet: Antibodies against BRD4 and c-Myc were purchased from Affinity Biosciences (DF2905, Liyang, China) and Proteintech (67447-1-Ig, Wuhan, China), respectively.

Techniques: Knockdown, In Vivo, Comparison, Imaging, Inhibition, Quantitative RT-PCR, Expressing, Western Blot, Control

BRD4 expression is elevated in the osteoporotic patients and animals. (A) Heatmap illustrating expression profiles of BRD gene family ( BRDT , BRD3 , BRD2 and BRD4 ) in the femoral head of normal and osteoporosis patients. (B) The expression levels of BRDT , BRD2 , BRD3 and BRD4 obtained from GSE230665 were quantified. (C) Violin plot presented the BRD4 expression in human bone marrow monocyte lineage cells (DISCO). (D) Violin plot presented the Brd4 expression in mouse bone marrow mesenchymal lineage cells, endothelial cells, mural cells and monocyte lineage cells ( GSE145477 ). (E) qRT‐PCR analysis reveals differential BRD4 mRNA expression in distal femur specimens from patients with varying bone mineral densities (BMD), classified as normal ( n = 7), osteopenia ( n = 7) and osteoporosis ( n = 6). (F) Correlation analysis between the BRD4 mRNA expression and the BMD measurements at the right femur (RF‐BMD) and the lumbar spine (LS‐BMD). (G) Representative images of H&E staining of distal femur bone of sham group and OVX group. (H) BMD and fat cells density of distal femur bone of sham group and OVX group ( n = 4). (I and J) Immunoblotting analysis of the Brd4 protein expression in the femur of 5‐month‐old sham‐operated or OVX mice ( n = 4). (K and L) Representative images of immunohistochemistry staining of Brd4 in the femur metaphysis, with the quantification of Brd4 + cells ( n = 3). (M) Uniform Manifold Approximation and Projection identified 10‐cell clusters in the bone marrow cells of osteoarthritis and osteoporosis patients. Each cluster is represented by a different colour. (N) Violin plot showing the elevated BRD4 expression in bone marrow cells of osteoporosis patients, compared with osteoarthritis patients. Comparisons in (F) were conducted using simple linear regression. Comparisons in the others were conducted by Student's t ‐test, two‐tailed. * p < .05, ** p < .01, **** p < .0001, n.s., not significant.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: BRD4 expression is elevated in the osteoporotic patients and animals. (A) Heatmap illustrating expression profiles of BRD gene family ( BRDT , BRD3 , BRD2 and BRD4 ) in the femoral head of normal and osteoporosis patients. (B) The expression levels of BRDT , BRD2 , BRD3 and BRD4 obtained from GSE230665 were quantified. (C) Violin plot presented the BRD4 expression in human bone marrow monocyte lineage cells (DISCO). (D) Violin plot presented the Brd4 expression in mouse bone marrow mesenchymal lineage cells, endothelial cells, mural cells and monocyte lineage cells ( GSE145477 ). (E) qRT‐PCR analysis reveals differential BRD4 mRNA expression in distal femur specimens from patients with varying bone mineral densities (BMD), classified as normal ( n = 7), osteopenia ( n = 7) and osteoporosis ( n = 6). (F) Correlation analysis between the BRD4 mRNA expression and the BMD measurements at the right femur (RF‐BMD) and the lumbar spine (LS‐BMD). (G) Representative images of H&E staining of distal femur bone of sham group and OVX group. (H) BMD and fat cells density of distal femur bone of sham group and OVX group ( n = 4). (I and J) Immunoblotting analysis of the Brd4 protein expression in the femur of 5‐month‐old sham‐operated or OVX mice ( n = 4). (K and L) Representative images of immunohistochemistry staining of Brd4 in the femur metaphysis, with the quantification of Brd4 + cells ( n = 3). (M) Uniform Manifold Approximation and Projection identified 10‐cell clusters in the bone marrow cells of osteoarthritis and osteoporosis patients. Each cluster is represented by a different colour. (N) Violin plot showing the elevated BRD4 expression in bone marrow cells of osteoporosis patients, compared with osteoarthritis patients. Comparisons in (F) were conducted using simple linear regression. Comparisons in the others were conducted by Student's t ‐test, two‐tailed. * p < .05, ** p < .01, **** p < .0001, n.s., not significant.

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with primary antibody against Brd4 (BETHYL; A700‐004, 1:1000), Nfatc1 (Cell Signaling Technology; 14074, 1:1000), GAPDH (Cell Signaling Technology; 2118, 1:1000), Ctsk (Abcam; ab19027, 1:1000), c‐Fos (Abcam; ab222699, 1:500), Acp5 (Abcam; ab235448, 1:500) and Slc9b2 (Immunoway; YN8225, 1:1000).

Techniques: Expressing, Quantitative RT-PCR, Staining, Western Blot, Immunohistochemistry, Two Tailed Test

Brd4 regulates osteoclastogenesis via glycolysis. (A) Validation of Brd4 degradation by dBET6 in Raw264.7 cells with immunoblotting analysis (top). Assessment of cell viability of Raw264.7 after 24 h treatment with various concentrations of dBET6 (bottom) ( n = 3). (B) Representative images of TRAP staining of Raw264.7 cells stimulated with RANKL and M‐CSF in the presence or absence of different concentrations of dBET6 (left), with quantitative analysis of the number and size of TRAP‐positive multinuclear cells per view (right) ( n = 5). (C) Representative images of F‐actin ring formation of Raw264.7 stimulated with RANKL and M‐CSF in the presence or absence of dBET6 at the indicated concentrations (left), with quantitative analysis of the number and size of F‐actin rings per view (right) ( n = 5). (D) qRT‐PCR detection of OC differentiation markers ( Nfatc1 and Ctsk ) in the Raw264.7 cells stimulated with RANKL and M‐CSF in the presence or absence of dBET6 at the indicated concentrations ( n = 3). (E) Seahorse analysis of extracellular acidification rate (ECAR) (left) and glycolysis (right) in the OC treated with different dBET6 concentrations for 24 h. Glu, glucose; Oligo, oligomycin; 2‐DG, 2‐deoxyglucose. (F) Representative images of TRAP staining (left) and quantification analysis (right) of BMMs from 8‐week‐old Lyz2‐Cre; Brd4 f/f mice and littermate control mice after 5 days of OC induction ( n = 3). (G) Representative images of F‐actin ring formation (left) and quantification analysis (right) of BMMs from 8‐week‐old Lyz2‐Cre; Brd4 f/f mice and littermate control mice after 5 days of OC induction ( n = 3). (H) Representative images of bone resorption pits on the bone slices stained with toluidine blue (left), with quantification of the pit area and number using Image J software (right) ( n = 3). (I) Seahorse analysis of extracellular acidification rate (ECAR) (left) and glycolysis (right) BMMs from Lyz2‐Cre; Brd4 f/f mice and littermate control mice after 5 days of OC induction. (J) Immunoblotting analysis of Nfatc1, c‐Fos and Ctsk in BMMs‐derived OC of Brd4 f/f ( WT ) and Lyz2‐Cre; Brd4 f/f ( cKO Lyz2 ) mice. (K) qRT‐PCR analysis of Nfatc1and Ctsk mRNA expression in BMMs treated with or without RANKL ( n = 3). Comparisons in (K) are conducted by one‐way ANOVA analyses. Comparisons in the others are conducted by Student's t ‐test, two‐tailed. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: Brd4 regulates osteoclastogenesis via glycolysis. (A) Validation of Brd4 degradation by dBET6 in Raw264.7 cells with immunoblotting analysis (top). Assessment of cell viability of Raw264.7 after 24 h treatment with various concentrations of dBET6 (bottom) ( n = 3). (B) Representative images of TRAP staining of Raw264.7 cells stimulated with RANKL and M‐CSF in the presence or absence of different concentrations of dBET6 (left), with quantitative analysis of the number and size of TRAP‐positive multinuclear cells per view (right) ( n = 5). (C) Representative images of F‐actin ring formation of Raw264.7 stimulated with RANKL and M‐CSF in the presence or absence of dBET6 at the indicated concentrations (left), with quantitative analysis of the number and size of F‐actin rings per view (right) ( n = 5). (D) qRT‐PCR detection of OC differentiation markers ( Nfatc1 and Ctsk ) in the Raw264.7 cells stimulated with RANKL and M‐CSF in the presence or absence of dBET6 at the indicated concentrations ( n = 3). (E) Seahorse analysis of extracellular acidification rate (ECAR) (left) and glycolysis (right) in the OC treated with different dBET6 concentrations for 24 h. Glu, glucose; Oligo, oligomycin; 2‐DG, 2‐deoxyglucose. (F) Representative images of TRAP staining (left) and quantification analysis (right) of BMMs from 8‐week‐old Lyz2‐Cre; Brd4 f/f mice and littermate control mice after 5 days of OC induction ( n = 3). (G) Representative images of F‐actin ring formation (left) and quantification analysis (right) of BMMs from 8‐week‐old Lyz2‐Cre; Brd4 f/f mice and littermate control mice after 5 days of OC induction ( n = 3). (H) Representative images of bone resorption pits on the bone slices stained with toluidine blue (left), with quantification of the pit area and number using Image J software (right) ( n = 3). (I) Seahorse analysis of extracellular acidification rate (ECAR) (left) and glycolysis (right) BMMs from Lyz2‐Cre; Brd4 f/f mice and littermate control mice after 5 days of OC induction. (J) Immunoblotting analysis of Nfatc1, c‐Fos and Ctsk in BMMs‐derived OC of Brd4 f/f ( WT ) and Lyz2‐Cre; Brd4 f/f ( cKO Lyz2 ) mice. (K) qRT‐PCR analysis of Nfatc1and Ctsk mRNA expression in BMMs treated with or without RANKL ( n = 3). Comparisons in (K) are conducted by one‐way ANOVA analyses. Comparisons in the others are conducted by Student's t ‐test, two‐tailed. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Techniques: Biomarker Discovery, Western Blot, Staining, Quantitative RT-PCR, Control, Software, Derivative Assay, Expressing, Two Tailed Test

Depletion of Brd4 in myeloid OC precursors protects mice from pathologic bone loss. (A) Representative micro‐CT images of distal femur bone of 12‐week‐old Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. (B) Bone parameters (BV/TV, Tb. Th, Tb. N and Tb. Sp) of the distal femur of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice induced by sham‐operated or OVX as analysed by micro‐CT ( n = 6). OVX/sham ratios were calculated for each genotype to illustrate genotype‐dependent differences. (C) Representative images of TRAP staining in the femur sections of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. The TRAP‐stained OC were denoted by the red arrow. (D) OC.N/BPm (OC number per bone parameter) and OC.S/BS (OC surface per bone surface) in C were quantified ( n = 6). (E) Representative micro‐CT images of trabecular bone in the distal femur of 12‐week‐old Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice treated by LPS or PBS for 8 days. (F and G) Bone parameters (BV/TV, Tb. Th, Tb. N and Tb. Sp) in the distal femur of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice treated by LPS or control PBS for 8 days ( n = 5). (H) Representative images of TRAP staining in the femur sections of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice treated by LPS or PBS for 8 days. (I) Quantification of the number of TRAP‐positive multinuclear cells and size of OC per field ( n = 6). All comparisons were conducted by one‐way ANOVA analyses. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: Depletion of Brd4 in myeloid OC precursors protects mice from pathologic bone loss. (A) Representative micro‐CT images of distal femur bone of 12‐week‐old Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. (B) Bone parameters (BV/TV, Tb. Th, Tb. N and Tb. Sp) of the distal femur of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice induced by sham‐operated or OVX as analysed by micro‐CT ( n = 6). OVX/sham ratios were calculated for each genotype to illustrate genotype‐dependent differences. (C) Representative images of TRAP staining in the femur sections of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. The TRAP‐stained OC were denoted by the red arrow. (D) OC.N/BPm (OC number per bone parameter) and OC.S/BS (OC surface per bone surface) in C were quantified ( n = 6). (E) Representative micro‐CT images of trabecular bone in the distal femur of 12‐week‐old Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice treated by LPS or PBS for 8 days. (F and G) Bone parameters (BV/TV, Tb. Th, Tb. N and Tb. Sp) in the distal femur of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice treated by LPS or control PBS for 8 days ( n = 5). (H) Representative images of TRAP staining in the femur sections of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice treated by LPS or PBS for 8 days. (I) Quantification of the number of TRAP‐positive multinuclear cells and size of OC per field ( n = 6). All comparisons were conducted by one‐way ANOVA analyses. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Techniques: Micro-CT, Staining, Control

Depletion of Brd4 in OC protects mice from pathologic bone loss by inhibiting OC activity. (A) Representative micro‐CT images of the distal femur of 12‐week‐old Brd4 f/f and Ctsk‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. (B) Bone parameters (BV/TV, Tb. Th, Tb. N and Tb. Sp) in the distal femur of Brd4 f/f and Ctsk‐Cre; Brd4 f/f mice subjected to sham‐operated or OVX ( n = 5). (C) Representative images of TRAP staining in the femur sections of Brd4 f/f and Ctsk‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. The TRAP‐stained OC were denoted by the red arrow. (D) The TRAP‐positive OC were quantified based on OC.N/BPm and OC.S/BS ( n = 5). (E) Representative images of TRAP staining (left) and quantification analysis (right) of BMMs from 8‐week‐old Ctsk‐Cre; Brd4 f/f mice and their control littermate after 5 days of OC induction ( n = 3). (F) Representative images of bone resorption pits on the bone slices stained with toluidine blue (left), with quantification of the pit area and number using Image J software (right) ( n = 3). Comparisons in panels (E and F) were conducted by Student's t ‐test, two‐tailed; in panels (B and D), by one‐way ANOVA analyses. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: Depletion of Brd4 in OC protects mice from pathologic bone loss by inhibiting OC activity. (A) Representative micro‐CT images of the distal femur of 12‐week‐old Brd4 f/f and Ctsk‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. (B) Bone parameters (BV/TV, Tb. Th, Tb. N and Tb. Sp) in the distal femur of Brd4 f/f and Ctsk‐Cre; Brd4 f/f mice subjected to sham‐operated or OVX ( n = 5). (C) Representative images of TRAP staining in the femur sections of Brd4 f/f and Ctsk‐Cre; Brd4 f/f mice induced by sham‐operated or OVX. The TRAP‐stained OC were denoted by the red arrow. (D) The TRAP‐positive OC were quantified based on OC.N/BPm and OC.S/BS ( n = 5). (E) Representative images of TRAP staining (left) and quantification analysis (right) of BMMs from 8‐week‐old Ctsk‐Cre; Brd4 f/f mice and their control littermate after 5 days of OC induction ( n = 3). (F) Representative images of bone resorption pits on the bone slices stained with toluidine blue (left), with quantification of the pit area and number using Image J software (right) ( n = 3). Comparisons in panels (E and F) were conducted by Student's t ‐test, two‐tailed; in panels (B and D), by one‐way ANOVA analyses. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Techniques: Activity Assay, Micro-CT, Staining, Control, Software, Two Tailed Test

Slc9b2 is required for Brd4‐mediated osteoclastogenesis. (A) Schematic diagram illustrating the transcriptomic analysis of differentially expressed genes (DEGs) in BMMs‐derived OC of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice. (B) Volcano plots highlighting the up‐regulated and down‐regulated DEGs in the BMMs‐derived OC of Lyz2‐Cre; Brd4 f/f group and Brd4 f/f mice. (C) Heat map showcasing the top 30 DEGs in BMM‐derived OCs, with triplicate data for each group. (D) KEGG enrichment analysis identifying the top 10 signalling pathways. (E) Immunoblotting analysis of the protein expression levels of Nfatc1 and Slc9b2 in BMMs treated with OC induction and 2‐DG or not. (F) qRT‐PCR detection of Slc9b2 mRNA expression level in BMMs of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with or without OC induction ( n = 3). (G and H) Immunoblotting analysis of the express ion of Slc9b2 and Nfatc1 in BMMs of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with OC induction or not (H), with quantitative analysis of protein expression levels of Slc9b2 G) ( n = 3). (I and J) BMMs from Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with OC induction were infected with lentivirus expressing vehicle control (Lv‐NC) or Slc9b2 (Lv‐ Slc9b2) for 24 h, followed by immunoblotting to detect Slc9b2 and Nfatc1 (I), with quantitative analysis of protein expression levels of Slc9b2 (J) ( n = 3). (K) qRT‐PCR detection of Slc9b2 mRNA expression level in BMMs of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with or without OC induction, after infected with Lv‐NC or Lv‐Slc9b2 for 24 h ( n = 3). (L) qRT‐PCR detection of the mRNA expression levels of Nfatc1 and Ctsk in BMMs‐derived OC from Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice ( n = 3). (M) Representative images of TRAP‐stained cells in BMM‐derived OCs from Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice infected with Lv‐NC or Lv‐Slc9b2 (top); Quantitative analysis of the number and size of TRAP‐positive multinuclear cells (bottom) (n = 3). (N) Representative images of F‐actin ring formation in BMM‐derived OCs from Lv‐NC and Lv‐Slc9b2 (top); quantitative analysis of the size and number of F‐actin rings per view (bottom) ( n = 3). All comparisons were conducted by one‐way ANOVA analyses. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: Slc9b2 is required for Brd4‐mediated osteoclastogenesis. (A) Schematic diagram illustrating the transcriptomic analysis of differentially expressed genes (DEGs) in BMMs‐derived OC of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice. (B) Volcano plots highlighting the up‐regulated and down‐regulated DEGs in the BMMs‐derived OC of Lyz2‐Cre; Brd4 f/f group and Brd4 f/f mice. (C) Heat map showcasing the top 30 DEGs in BMM‐derived OCs, with triplicate data for each group. (D) KEGG enrichment analysis identifying the top 10 signalling pathways. (E) Immunoblotting analysis of the protein expression levels of Nfatc1 and Slc9b2 in BMMs treated with OC induction and 2‐DG or not. (F) qRT‐PCR detection of Slc9b2 mRNA expression level in BMMs of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with or without OC induction ( n = 3). (G and H) Immunoblotting analysis of the express ion of Slc9b2 and Nfatc1 in BMMs of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with OC induction or not (H), with quantitative analysis of protein expression levels of Slc9b2 G) ( n = 3). (I and J) BMMs from Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with OC induction were infected with lentivirus expressing vehicle control (Lv‐NC) or Slc9b2 (Lv‐ Slc9b2) for 24 h, followed by immunoblotting to detect Slc9b2 and Nfatc1 (I), with quantitative analysis of protein expression levels of Slc9b2 (J) ( n = 3). (K) qRT‐PCR detection of Slc9b2 mRNA expression level in BMMs of Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice with or without OC induction, after infected with Lv‐NC or Lv‐Slc9b2 for 24 h ( n = 3). (L) qRT‐PCR detection of the mRNA expression levels of Nfatc1 and Ctsk in BMMs‐derived OC from Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice ( n = 3). (M) Representative images of TRAP‐stained cells in BMM‐derived OCs from Brd4 f/f and Lyz2‐Cre; Brd4 f/f mice infected with Lv‐NC or Lv‐Slc9b2 (top); Quantitative analysis of the number and size of TRAP‐positive multinuclear cells (bottom) (n = 3). (N) Representative images of F‐actin ring formation in BMM‐derived OCs from Lv‐NC and Lv‐Slc9b2 (top); quantitative analysis of the size and number of F‐actin rings per view (bottom) ( n = 3). All comparisons were conducted by one‐way ANOVA analyses. * p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s., not significant.

Techniques: Derivative Assay, Western Blot, Expressing, Quantitative RT-PCR, Infection, Control, Staining

dBET6@PSLs prevents pathological bone loss via regulating Slc9b2. (A) Schematic showing the in vivo therapeutic approach administrated to 12‐week‐old WT mice. The treatment protocol involved intramedullary injections of dBET6@PSLs, followed by intraperitoneal administration of either LPS or PBS and the late pathological analysis. (B) Representative micro‐CT photographs depict the skeletal alteration in 12‐week‐old WT mice treated with 50 or 200 nM dBET6@PSLs with or without LPS induction. (C and D) Micro‐CT analysis (BMD, BV/TV of cortical, BV/TV, Tb.Th, Tb.N and Tb.Sp) of the distal femur in (B) ( n = 3). (E) Representative images of TRAP‐stained cells in the femur sections of 12‐week‐old WT mice treated with 50 or 200 nM dBET6@PSLs with or without LPS induction. (F) The TRAP‐positive OC indicated by the red arrow were quantified with respect to OC.N/BPm and OC.S/BS ( n = 3). (G) Representative immunofluorescence images of the distal femur stained with Brd4 (green) and Slc9b2 (red) in the 12‐week‐old WT mice treated with 50 or 200 nM dBET6@PSLs with or without LPS induction. (H, I) Quantitative analysis of Brd4 and Slc9b2 mean fluorescence intensity was performed on multiple randomly selected fields per sample ( n = 3). All comparisons were conducted by Student's t ‐test, two‐tailed. * p < .05, ** p < .01, *** p < .001, n.s., not significant.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: dBET6@PSLs prevents pathological bone loss via regulating Slc9b2. (A) Schematic showing the in vivo therapeutic approach administrated to 12‐week‐old WT mice. The treatment protocol involved intramedullary injections of dBET6@PSLs, followed by intraperitoneal administration of either LPS or PBS and the late pathological analysis. (B) Representative micro‐CT photographs depict the skeletal alteration in 12‐week‐old WT mice treated with 50 or 200 nM dBET6@PSLs with or without LPS induction. (C and D) Micro‐CT analysis (BMD, BV/TV of cortical, BV/TV, Tb.Th, Tb.N and Tb.Sp) of the distal femur in (B) ( n = 3). (E) Representative images of TRAP‐stained cells in the femur sections of 12‐week‐old WT mice treated with 50 or 200 nM dBET6@PSLs with or without LPS induction. (F) The TRAP‐positive OC indicated by the red arrow were quantified with respect to OC.N/BPm and OC.S/BS ( n = 3). (G) Representative immunofluorescence images of the distal femur stained with Brd4 (green) and Slc9b2 (red) in the 12‐week‐old WT mice treated with 50 or 200 nM dBET6@PSLs with or without LPS induction. (H, I) Quantitative analysis of Brd4 and Slc9b2 mean fluorescence intensity was performed on multiple randomly selected fields per sample ( n = 3). All comparisons were conducted by Student's t ‐test, two‐tailed. * p < .05, ** p < .01, *** p < .001, n.s., not significant.

Techniques: In Vivo, Micro-CT, Staining, Immunofluorescence, Fluorescence, Two Tailed Test

Brd4 regulates bone metabolism through Slc9b2 suppression: a targeted therapeutic approach for osteoporosis. Elevated Brd4 expression is strongly correlated with osteoporosis, primarily by promoting osteoclastogenesis. Mechanistically, Brd4 is crucial role in regulating glycolysis, a prerequisite for osteoclastogenesis (left). In contrast, the loss of Brd4 has been shown to increase basal bone mass and prevent pathological bone loss induced by OVX or LPS, through the suppression of OC markers, particularly Slc9b2. Targeting Brd4 with PROTACs loaded on PSLs (dBET6@PSLs) significantly inhibited osteoclastogenesis and alleviated pathological bone loss. These findings suggest that Brd4 inhibition could be a promising therapeutic strategy for preventing pathological bone loss, including osteoporosis.

Journal: Clinical and Translational Medicine

Article Title: Loss of Brd4 alleviates pathological bone loss via Slc9b2 suppression in osteoclastogenesis

doi: 10.1002/ctm2.70496

Figure Lengend Snippet: Brd4 regulates bone metabolism through Slc9b2 suppression: a targeted therapeutic approach for osteoporosis. Elevated Brd4 expression is strongly correlated with osteoporosis, primarily by promoting osteoclastogenesis. Mechanistically, Brd4 is crucial role in regulating glycolysis, a prerequisite for osteoclastogenesis (left). In contrast, the loss of Brd4 has been shown to increase basal bone mass and prevent pathological bone loss induced by OVX or LPS, through the suppression of OC markers, particularly Slc9b2. Targeting Brd4 with PROTACs loaded on PSLs (dBET6@PSLs) significantly inhibited osteoclastogenesis and alleviated pathological bone loss. These findings suggest that Brd4 inhibition could be a promising therapeutic strategy for preventing pathological bone loss, including osteoporosis.

Techniques: Expressing, Inhibition

Review

Structured Review

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1) Product Images from "E2F1 K117 methylation by SETD6 disrupts BRD4–E2F1 binding and modulates E2F1 chromatin binding and gene regulation in prostate cancer cells"

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