Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: O-GlcNAcylation of BRD4 inhibited NF-κB p65-mediated transcription of pro-inflammatory cytokines. (A)&(B) The expression of BRD4 in OGD-exposed cardiomyocytes was detected by RT-qPCR and Western blotting. H9C2 and AC-16 cells were transfected with shBRD4, and then subjected to OGD. (C)&(D) RT-qPCR and Western blotting analysis of BRD4 mRNA and protein levels. (E)&(F) The mRNA levels and concentrations of TNF-α, IL-1β, and IL-6 were determined by RT-qPCR and ELISA. (G) The binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters was confirmed by dual-luciferase reporter assay. (H)&(I) Co-IP assay verified the exogenous and endogenous interplay between OGT and BRD4 proteins. (J) O-GlcNAcylation of BRD4 protein in OGD-stimulated cardiomyocytes was evaluated. (K) YinOYang database predicated the potential O-GlcNAc sites on BRD4. OGD-challenged H9C2 and AC-16 cells were transfected with BRD4 WT plasmid or BRD4 plasmids with mutant O-GlcNAc sites (BRD4-S484R, BRD4-S784R, and BRD4-T1212R). (L) O-GlcNAcylation of BRD4 protein in H9C2 and AC-16 cells was detected. (M) Concentrations of TNF-α, IL-1β, and IL-6 were detected by ELISA. (N) The interaction between NF-κB p65 and TNF-α, IL-1β, and IL-6 promoters was validated by dual-luciferase reporter assay. n=3 for A-N. Student's t test (for A, B) and one-way ANOVA (for C-G, M, N) were performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Binding Assay, Luciferase, Reporter Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Mutagenesis

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: ENPP3 contributed to inflammation by inhibiting O-GlcNAcylation of BRD4. H9C2 and AC-16 cells were transfected with shENPP3, followed by exposure to OGD. (A) ENPP3 and BRD4 protein levels were measured by Western blotting. (B) The O-GlcNAc level of BRD4 protein was assessed. (C) The production of TNF-α, IL-1β, and IL-6 was determined by ELISA. (D) Dual-luciferase reporter assay evaluated the binding of NF-κB p65 to TNF-α, IL-1β, and IL-6 promoters. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Binding Assay

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: SRSF1/ENPP3 axis suppressed BRD4 O-GlcNAcylation to promote inflammation in CME. The OGD-stimulated cardiomyocytes were transfected with shSRSF1, ENPP3 overexpression plasmid, or a combination of them. (A) ENPP3 mRNA and lncRNA ENPP3 expression levels were detected by RT-qPCR. (B) The protein abundance of ENPP3 and BRD4 was assessed by Western blotting. (C) The O-GlcNAc level of BRD4 was determined. (D) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=3 for A-D. One-way ANOVA was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Transfection, Over Expression, Plasmid Preparation, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Theranostics

Article Title: Divergent splicing factor SRSF1 signaling promotes inflammation post-CME: the SRSF1/ENPP3 axis acts via inhibition of BRD4 O-GlcNAcylation to enhance NF-κB activation and accelerate heart failure

doi: 10.7150/thno.115402

Figure Lengend Snippet: Myocardium-specific SRSF1 knockout alleviated CME-induced inflammation via inactivation of the ENPP3/BRD4/NF-κB pathway. SRSF1 flox/flox and SRSF1-KO rats were injected with microspheres into the left ventricle to induce CME. (A) LVEF, LVFS, LVEDd, and CO were detected to evaluate cardiac function. (B) The serum cTnl level in different groups was measured by ELISA. (C) Pathological alterations in myocardial tissues were observed by HE staining (scale bar = 100 μm). (D) Myocardial infarct size was measured by HBFP staining (scale bar = 100 μm). (E) SRSF1, ENPP3, and BRD4 expression in myocardial tissues was evaluated by immunohistochemical staining (scale bar = 100 μm). (F) The protein abundance of SRSF1, ENPP3, BRD4, p65, and O-GlcNAcylation of BRD4 was detected by Western blotting or Co-IP, respectively. (G) ELISA was carried out to measure TNF-α, IL-1β, and IL-6 concentrations. n=6 for A-G. ANOVA for repeated measurement (for A, B), and one-way ANOVA (for F, G) was performed to analyze data. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The sections received overnight incubation with primary antibodies SRSF1 (12929-2-AP, 1:50, Proteintech, Wuhan, China), ENPP3 (A05615, 1:100, Boster, CA, USA), or BRD4 (M00123, 1:50, Boster) at 4 °C.

Techniques: Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Immunohistochemical staining, Quantitative Proteomics, Western Blot, Co-Immunoprecipitation Assay

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:

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

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

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

Journal: Genes

Article Title: Systems Genetics Reveals the Gene Regulatory Mechanisms of Arrb2 in the Development of Autism Spectrum Disorders.

doi: 10.3390/genes16050605

Figure Lengend Snippet: Figure 6. Phenome-wide association study (PheWAS) analysis of genetic variations in strong can- didate genes regulated by Arrb2. Scatter plots show the PheWAS results for (A) Dnmt3a, (B) Myh9, (C) Ccdc88a, (D) Dnmt1, (E) Becn1, (F) Gng2, (G) Psmb6, and (H) Brd4. Each scatter represents a phenotype, and the x-axis coordinates of the scatter are randomly distributed. The y-axis represents the association significance between each gene and central nervous system-related traits in BXD mice, with the red dotted line marking the statistical significance threshold (–log10(p) = 3).

Article Snippet: The PVDF membrane was incubated at 4 ◦C overnight with primary antibodies, including mouse anti GAPDH (HRP-conjugated, 1:10,000, Proteintech, 60,004, Wuhan, Hubei, China), rabbit anti β-Arrestin2 (1:1000, Cell Signaling Technology, 3857, Danvers, Massachusetts, USA), rabbit anti Dnmt1 (1:2000, HuaBio, ET1702-77, Hangzhou, Zhejiang, China), rabbit anti Myh9 (1:2000, Abclonal, A0173, Wuhan, Hubei, China), rabbit anti Brd4 (1:1000, HuaBio, HA722785, Hangzhou, Zhejiang, China), rabbit anti p-Synapsin I (S9) (1:1000, HuaBio, ET1611-26, Hangzhou, Zhejiang, China), rabbit anti Synapsin I (1:1000, abcam, ab64581, Cambridge, UK) and rabbit anti p-PKA α/β/γ (catalytic subunit) (T197) (1:1000, HuaBio, HA721864, Hangzhou, Zhejiang, China).

Techniques:

Journal: Genes

Article Title: Systems Genetics Reveals the Gene Regulatory Mechanisms of Arrb2 in the Development of Autism Spectrum Disorders.

doi: 10.3390/genes16050605

Figure Lengend Snippet: Figure 7. Construction of Arrb2−/−mice and analysis of mRNA and protein levels of downstream candidate genes in the hippocampus. (A) Schematic diagram of the construction of Arrb2−/−mice. (B) qRT-PCR analysis of Arrb2 mRNA in the hippocampus of WT and Arrb2−/−mice. (C) Western blot analysis of Arrb2 protein in the hippocampus of WT and Arrb2−/−mice. (D–K) mRNA levels of downstream candidate genes regulated by Arrb2 in the hippocampus of WT and Arrb2−/−mice measured by qRT-PCR. (L–O) Western blot analysis of Myh9, Dnmt1, and Brd4 protein in the hippocampus of WT and Arrb2−/−mice. ns: p > 0.05, * p < 0.05, *** p < 0.001, unpaired two-tailed Student’s t-test, n = 6 per group.

Article Snippet: The PVDF membrane was incubated at 4 ◦C overnight with primary antibodies, including mouse anti GAPDH (HRP-conjugated, 1:10,000, Proteintech, 60,004, Wuhan, Hubei, China), rabbit anti β-Arrestin2 (1:1000, Cell Signaling Technology, 3857, Danvers, Massachusetts, USA), rabbit anti Dnmt1 (1:2000, HuaBio, ET1702-77, Hangzhou, Zhejiang, China), rabbit anti Myh9 (1:2000, Abclonal, A0173, Wuhan, Hubei, China), rabbit anti Brd4 (1:1000, HuaBio, HA722785, Hangzhou, Zhejiang, China), rabbit anti p-Synapsin I (S9) (1:1000, HuaBio, ET1611-26, Hangzhou, Zhejiang, China), rabbit anti Synapsin I (1:1000, abcam, ab64581, Cambridge, UK) and rabbit anti p-PKA α/β/γ (catalytic subunit) (T197) (1:1000, HuaBio, HA721864, Hangzhou, Zhejiang, China).

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

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: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

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

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.

Article Snippet: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

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

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.

Article Snippet: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

Techniques: Micro-CT, Staining, Control

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.

Article Snippet: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

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

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.

Article Snippet: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

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

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.

Article Snippet: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

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

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.

Article Snippet: For IF staining, frozen sections were incubated overnight at 4°C with primary antibodies against Brd4 (BETHYL; A700‐004, 1:200), Runx2 (Abcam; ab236639, 1:100), Sox9 (#82630; Cell Signaling Technology; 1:100), F4/80 (Servicebio; GB113373 , 1:400), Slc9b2 (Immunoway; YN8225, 1:100) and Ctsk (Abcam; ab19027, 1:100), respectively.

Techniques: Expressing, Inhibition