Journal: iScience

Article Title: Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

doi: 10.1016/j.isci.2023.107022

Figure Lengend Snippet:

Article Snippet: The following day, the membranes were washed three times with TBS-T buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 10 min each time, and then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit (1:10000, ab6721, abcam) or goat anti-mouse IgG (1:10000, G1214, Servicebio) secondary antibody, respectively.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Staining, Enzyme-linked Immunosorbent Assay, Software, Microscopy

Journal: Poultry Science

Article Title: Immunocastration with recombinant GnRH6-CRM197 fusion protein improves reproductive suppression and meat tenderness in roosters

doi: 10.1016/j.psj.2026.107046

Figure Lengend Snippet: SDS-PAGE analysis of expression of pET21a-GnRH6-CRM197 in E. coli. BL21. Lane 1, standard molecular weight marker; Lane 2, non-induced recombinant bacteria; Lane 3, recombinant bacteria induced after 12 h.

Article Snippet: The GnRH6-CRM197 recombinant protein was separated on a 12.5% SDS polyacrylamide gel at 120 V. It was then transferred to a 0.45μm polyvinylidene fluoride (PVDF) membrane (Pulilai, Beijing, China) at 250 mA for 1 hour using a transfer system (BVT-2, Servicebio, Wuhan, China).

Techniques: SDS Page, Expressing, Molecular Weight, Marker, Recombinant, Bacteria

Journal: Poultry Science

Article Title: Immunocastration with recombinant GnRH6-CRM197 fusion protein improves reproductive suppression and meat tenderness in roosters

doi: 10.1016/j.psj.2026.107046

Figure Lengend Snippet: WB analysis of recombinant GnRH6-CRM197 protein. Lane 1, standard molecular weight marker; Lane 2, recombinant GnRH6-CRM197 fusion protein reacted with rabbit polyclonal to GnRH (dilution 1:2000) .

Article Snippet: The GnRH6-CRM197 recombinant protein was separated on a 12.5% SDS polyacrylamide gel at 120 V. It was then transferred to a 0.45μm polyvinylidene fluoride (PVDF) membrane (Pulilai, Beijing, China) at 250 mA for 1 hour using a transfer system (BVT-2, Servicebio, Wuhan, China).

Techniques: Recombinant, Molecular Weight, Marker

Journal: Poultry Science

Article Title: Immunocastration with recombinant GnRH6-CRM197 fusion protein improves reproductive suppression and meat tenderness in roosters

doi: 10.1016/j.psj.2026.107046

Figure Lengend Snippet: The effect of recombinant anti-GnRH6-CRM197 on serum anti-GnRH antibody of roosters. The arrow refers to the immune time; the first immunity of roosters is 4 W; * P < 0.05; ** P < 0.01 (n = 20 per group).

Article Snippet: The GnRH6-CRM197 recombinant protein was separated on a 12.5% SDS polyacrylamide gel at 120 V. It was then transferred to a 0.45μm polyvinylidene fluoride (PVDF) membrane (Pulilai, Beijing, China) at 250 mA for 1 hour using a transfer system (BVT-2, Servicebio, Wuhan, China).

Techniques: Recombinant

Journal: Poultry Science

Article Title: Immunocastration with recombinant GnRH6-CRM197 fusion protein improves reproductive suppression and meat tenderness in roosters

doi: 10.1016/j.psj.2026.107046

Figure Lengend Snippet: The effect of recombinant GnRH6-CRM197 on serum testosterone levels of roosters. * P < 0.05; ** P < 0.01 (n = 20 per group).

Article Snippet: The GnRH6-CRM197 recombinant protein was separated on a 12.5% SDS polyacrylamide gel at 120 V. It was then transferred to a 0.45μm polyvinylidene fluoride (PVDF) membrane (Pulilai, Beijing, China) at 250 mA for 1 hour using a transfer system (BVT-2, Servicebio, Wuhan, China).

Techniques: Recombinant

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