Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: (A) HepAD38, HepBHAe82, and HepBHAeΔx67 cells were induced for HBV production for 14 days. The expression of endogenous HMGB1 was detected by Western blot, β-actin served as a loading control. The association of HMGB1 with cccDNA was analyzed by ChIP-qPCR and presented in percentage (%) of input (mean ± SEM, n = 3). (B) HepG2-NTCP cells were infected with wt HBV or HBV Δ x for 10 days and subjected to the same analyses as aforementioned in (A). *p<0.05, **p<0.01.

Article Snippet: The Co-IP of HBx and endogenous HMGB1 protein were performed in cells transfected by FLAG-HBx plasmid using Anti-HMGB1/HMG1 Magnetic Beads Immunoprecipitation (IP) Kit (MB100930-T46, Sino Biologicals) followed by Western immunoblotting.

Techniques: Expressing, Western Blot, Infection

Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: (A) HepG2 cells were transfected with control vector or FLAG-HBx for 3 days. The expression of endogenous HMGB1 and transfected FLAG-HBx were detected by Western blot, β-actin served as a loading control. Co-immunoprecipitation of endogenous HMGB1 was performed, and the immunoprecipitated HMGB1 and FLAG-HBx were detected by Western blot. (B) Cells were transfected with (1) His-HMGB1 or (2) FLAG-HBx or (3) both for 3 days, followed by immunofluorescence microscopy analyses of His-HMGB1 (stained in red) and FLAG-HBx (stained in green), their colocalization was shown as bright yellow to orange signals. Cell nuclei were stained by DAPI (blue). Scale bar = 10 μm.

Article Snippet: The Co-IP of HBx and endogenous HMGB1 protein were performed in cells transfected by FLAG-HBx plasmid using Anti-HMGB1/HMG1 Magnetic Beads Immunoprecipitation (IP) Kit (MB100930-T46, Sino Biologicals) followed by Western immunoblotting.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Immunofluorescence, Microscopy, Staining

Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: (A) Assessment of the HepBHAeΔx67-shControl and HepBHAeΔx67-shHMGB1 cell lines. The knockdown of HMGB1 in HepBHAeΔx67-shHMGB1 cells was validated by Western blot, β-actin served as loading control. After 6-days induction, HBV total RNA was analyzed by Northern blot, cellular 28S and 18S rRNA served as loading control; HBV cytoplasmic core DNA was analyzed by Southern blot, mitochondrial (mt) DNA served as a loading control. After 14-days induction, total cellular Hirt DNA was heat-denatured, followed by EcoRI digestion, and then subjected to Southern blot analyses of HBV deproteinated rcDNA (DP-RC), linearized cccDNA, and mtDNA. (B) The relative levels of HBV cccDNA-dependent pC mRNA in HepBHAeΔx67-shControl and HepBHAeΔx67-shHMGB1 cells were analyzed by qPCR and normalized to cellular GAPDH and cccDNA (mean ± SEM, n = 3). (C) Supernatant HA-HBeAg was detected by CLIA (mean ± SEM, n = 3). ***p<0.001.

Article Snippet: The Co-IP of HBx and endogenous HMGB1 protein were performed in cells transfected by FLAG-HBx plasmid using Anti-HMGB1/HMG1 Magnetic Beads Immunoprecipitation (IP) Kit (MB100930-T46, Sino Biologicals) followed by Western immunoblotting.

Techniques: Western Blot, Northern Blot, Southern Blot

Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: HepG2-NTCP-shControl and HepG2-NTCP-shHMGB1 cells were infected by wt HBV at 500 vge/cell for 10 days. (A-B) The levels of HMGB1 protein expression were analyzed by Western blot. HBV pC mRNA and total HBV RNA were analyzed by qPCR and normalized to GAPDH mRNA and HBV cccDNA (mean ± SEM, n = 3). The enrichment of RNAPII pho-CTD (C), H3K27ac (D), and H3K27me3 (E) on cccDNA was analyzed by ChIP-qPCR as presented in percentage (%) of input (mean ± SEM, n = 3). **p<0.01, ***p<0.001.

Article Snippet: The Co-IP of HBx and endogenous HMGB1 protein were performed in cells transfected by FLAG-HBx plasmid using Anti-HMGB1/HMG1 Magnetic Beads Immunoprecipitation (IP) Kit (MB100930-T46, Sino Biologicals) followed by Western immunoblotting.

Techniques: Infection, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Hepatitis B virus X protein counteracts high mobility group box 1 protein-mediated epigenetic silencing of covalently closed circular DNA

doi: 10.1371/journal.ppat.1010576

Figure Lengend Snippet: Newly formed cccDNA is targeted by preexisting intranuclear host restriction factor HMGB1, which mediates epigenetic silencing of cccDNA minichromosome. Transcriptionally repressed cccDNA lacks active histone PTMs (represented by H3K27ac and H3K4me3) and enriched with repressive histone PTMs (represented by H3K27me3 and H3K9me3). Viral HBx protein counteracts HMGB1 to prevent its association with cccDNA and induce partial nuclear-cytoplasmic translocation of HMGB1, thereby conferring an active epigenetic state of cccDNA transcription.

Article Snippet: The Co-IP of HBx and endogenous HMGB1 protein were performed in cells transfected by FLAG-HBx plasmid using Anti-HMGB1/HMG1 Magnetic Beads Immunoprecipitation (IP) Kit (MB100930-T46, Sino Biologicals) followed by Western immunoblotting.

Techniques: Translocation Assay

Journal: Frontiers in Public Health

Article Title: Proton beam therapy induces protective immunity via HMGB1-dependent signaling

doi: 10.3389/fpubh.2025.1686678

Figure Lengend Snippet: The expression of proton-induced CRT and HMGB1. (A,C,D) Flow cytometry analysis of CRT at 24 h post-proton irradiation. (B,E,F) Flow cytometry analysis of CRT at 48 h post-proton irradiation. (G) The temporal characteristics of proton-induced CRT expression. (H–J) The expression of HMGB1 in the supernatant of cell culture medium 24 (I) and 48 (J) hours after proton irradiation. (H) The temporal characteristics of proton-induced HMGB1 expression. MFI means median fluorescence intensity. * means p < 0.05, ** means p < 0.01,*** means p < 0.001, ns means not significant.

Article Snippet: HMGB1 and Interferon-gamma (IFN-γ) in the mouse serum samples were measured by using HMGB1 kit (H257-1-2, Mouse HMGB1 ELISA Kit, NJJC Bioscience, Nanjing, China) and IFN-γ kit (#EK0375, Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Irradiation, Cell Culture, Fluorescence

Journal: Frontiers in Public Health

Article Title: Proton beam therapy induces protective immunity via HMGB1-dependent signaling

doi: 10.3389/fpubh.2025.1686678

Figure Lengend Snippet: Proton radiation stimulates immunogenic cell death. (A) Experimental schedule for proton-induced ICD evaluation. Primary tumor growth (B) and distal tumor growth (C) after the transplantation of different doses (3, 6, 12 Gy) proton-irradiated cells. Four weeks after tumor cell transplantation, spleens were harvested and analysed: immunohistochemical staining of T lymphocytes (D) and IFN- γ (G) infiltration in the spleen, and quantification by flow cytometry (E,F) . (H) The expression of HMGB1 in serum after the transplantation of proton-irradiated cells. * means p < 0.05, ** means p < 0.01,*** means p < 0.001, **** means p < 0.0001, ns means not significant.

Article Snippet: HMGB1 and Interferon-gamma (IFN-γ) in the mouse serum samples were measured by using HMGB1 kit (H257-1-2, Mouse HMGB1 ELISA Kit, NJJC Bioscience, Nanjing, China) and IFN-γ kit (#EK0375, Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Irradiation, Immunohistochemical staining, Staining, Flow Cytometry, Expressing

Journal: Frontiers in Public Health

Article Title: Proton beam therapy induces protective immunity via HMGB1-dependent signaling

doi: 10.3389/fpubh.2025.1686678

Figure Lengend Snippet: Verification of silencing of shCRT and shHMGB1 cell lines by proton irradiation. The mRNA expression level of CRT (A) and HMGB1 (B) after corresponding gene silencing. Surface expression of CRT (C) and secreted HMGB1 concentration (D) in CRT- or HMGB1-knockdown Colon-26 stable cell lines were measured after irradiation by flow cytometry and ELISA, respectively. * means p < 0.05, ** means p < 0.01,*** means p < 0.001, **** means p < 0.0001, ns means not significant.

Article Snippet: HMGB1 and Interferon-gamma (IFN-γ) in the mouse serum samples were measured by using HMGB1 kit (H257-1-2, Mouse HMGB1 ELISA Kit, NJJC Bioscience, Nanjing, China) and IFN-γ kit (#EK0375, Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Irradiation, Expressing, Concentration Assay, Knockdown, Stable Transfection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Public Health

Article Title: Proton beam therapy induces protective immunity via HMGB1-dependent signaling

doi: 10.3389/fpubh.2025.1686678

Figure Lengend Snippet: The role of Proton-induced CRT and HMGB1 in distal tumor colonization rejection. (A) Experimental schedule for CRT and HMGB1 evaluation. (B) Tumor growth after the transplantation of 12 Gy Proton-irradiated cells. The black dotted line indicates mean volume of non-irradiated treated tumor. The blue solid line indicates that the volume of the distal tumor is close to zero, meaning a state of complete immunity. The orange solid line indicates that the volume of the distal tumor is above zero yet remains below those of untreated controls, meaning a state of partial immune control. (C) The expression of HMGB1 in serum after the transplantation of 12 Gy Proton-irradiated cells. Ratio of CD4 positive T cell.

Article Snippet: HMGB1 and Interferon-gamma (IFN-γ) in the mouse serum samples were measured by using HMGB1 kit (H257-1-2, Mouse HMGB1 ELISA Kit, NJJC Bioscience, Nanjing, China) and IFN-γ kit (#EK0375, Boster Biological Technology, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Irradiation, Control, Expressing

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: Intramuscular injection of fr-HMGB1 or 3S accelerates muscle regeneration. (A) Muscle acute injury was induced by injection of Ctx in triceps muscles. Vehicle (PBS) or HMGB1 (fr-HMGB1 or ds-HMGB1) was injected at the same time as Ctx. Quantitative PCR of Pax7, MyoD, and Myogenin (Myog) mRNA in triceps was performed at day 5 after injury (fold increase vs. vehicle). The significance of the difference in gene expression between mice treated with HMGB1 (fr-HMGB1 or ds-HMGB1) and vehicle-treated mice was assessed with Mann-Whitney tests. *, P < 0.05; **, P < 0.01. n = 9 mice per group, three independent experiments. (B–I) Ctx was injected together with vehicle (PBS) or WT fr-HMGB1 (HMGB1) or 3S in TA and/or triceps muscles. Muscle regeneration was assessed at days 2, 5, 10, 15, and 20 after injury. (B) Quantitative PCR of Pax7, MyoD, and Myog mRNA in triceps at days 5, 10, 15, and 20 after injury (fold increase vs. uninjured muscles). n ≥ 5 mice per group, at least two independent experiments. (C) TA muscles, stained with EBD, at days 5 and 15 after injury. (D) EBD, laminin, and DAPI staining of TA muscle sections at days 2, 5, and 15 after injury. Bars, 50 µm. (E) Percentage of EBD-positive myofibers in regenerating TA muscles at day 5 after injury. n = 4 mice per group, two independent experiments. (F) CSA of fibers in TA muscles at days 5 and 15 after injury. n ≥ 5 mice per group, at least two independent experiments. (G) Quantification of myofibers with nuclei at the periphery (PNF) in TA muscles at day 15 after injury. n = 5 mice per group, two independent experiments. (H) CD31 and DAPI staining on TA muscle sections at day 15 after injury and relative quantification of CD31 + area. n = 3 mice per group of three independent experiments. Bars, 50 µm. (I) Tetanic force of TA muscles: uninjured (No Ctx), cardiotoxin-injured (Ctx), Ctx-injured treated with 3S (Ctx + 3S), at day 10 after injury. n = 5 mice per group, two independent experiments. Differences between groups in B and E–I were assessed with one-way ANOVA plus Dunnett’s post-test; data are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Injection, Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY, Staining

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: fr-HMGB1/3S promotes skeletal muscle regeneration by acting on satellite cells. (A) TA muscle acute injury was induced by injection of Ctx; vehicle (PBS) or WT fr-HMGB1 (HMGB1) or 3S was injected together with Ctx. Pax7 + cells were quantified in regenerating TA muscles at days 5 and 15 after injury. n = 3 mice per group, two independent experiments. (B) Ex vivo migration of primary mouse Pax7 + cells toward 40 nM HMGB1 (fully reduced) or 3S. Hepatocyte growth factor (HGF) was used as the positive control. n = 3 independent experiments. (C–E) Single myofibers isolated from mouse muscles were cultured in proliferation medium for 72 (C and D) or 16 h (E) with or without 40 nM fr-HMGB1 or 3S. Relative numbers of MyoD + and/or Myogenin + cells at 72 h (D) and of Pax7 + and/or MyoD + cells at 16 h (E) on single fibers. n = 3 mice, 10 fibers/mouse, three independent experiments. Bars, 50 µm. (F and G) Representative images of mouse primary myoblasts cultured for 48 h in differentiating medium, with or without 40 nM fr-HMGB1 or 3S, and stained with DAPI and anti–myosin heavy chain (MHC) antibody (F), and distribution of myofiber size (G). Bar, 100 µm. The differences between control and treatments are statistically significant (P < 0.0001, χ 2 test). Data are representative of two independent experiments with biological triplicates. Data are means ± SEM. Statistical significance was assessed with one-way ANOVA plus Dunnett’s post-test in A, B, D, and E. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Injection, Ex Vivo, Migration, Positive Control, Isolation, Cell Culture, Staining

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: fr-HMGB1/3S modulates macrophage polarization toward a tissue-healing phenotype. (A–D) TA muscles were injected with Ctx alone or Ctx plus WT fr-HMGB1 (HMGB1) or 3S. (A) Quantification of CD45 + cells isolated from injured muscles at days 1 ( n = 5 mice per group of three independent experiments) and 2 ( n = 3 mice per group of three independent experiments) after injury. (B and C) Representative immunofluorescence staining for CD68, CD86, and CD163 on sections of TA muscles at day 5 after injury (B) and quantification of CD68 + ( n = 3 mice per group), CD68 + CD86 + , and CD68 + CD163 + cells ( n = 5 mice per group) in sections of TA muscles (C), two independent experiments. Bars, 50 µm. (D) Quantitative PCR of IGF-1 mRNA in triceps at day 5 after injury. n = 9 mice per group of three independent experiments. (E and F) Mouse bone marrow–derived macrophages were cultured for 7 d in DMEM conditioned by L929 cells (enriched in CSF-1) and polarized for 3 d toward a proinflammatory phenotype by stimulation with IFNγ (50 ng/ml) with or without 40 nM fr-HMGB1 or 3S. Representative images of macrophages (E) and quantification of cells positive for iNOS, TNFα, TGFβ, and CD163 expression, analyzed by immunofluorescence staining (F; fold increase vs. control; at least three independent experiments). Bars, 50 µm. Data are means ± SEM. Statistical significance was assessed with one-way ANOVA plus Dunnett’s post-test in A, C, D, and F. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Injection, Isolation, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Derivative Assay, Cell Culture, Expressing

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: High expression of HMGB1 is required for optimal skeletal muscle regeneration . Muscle acute injury was induced by injection of Ctx in TA and/or triceps muscles of WT or Hmgb1 +/− mice, and regeneration was assessed at day 5 after injury. (A) H&E and immunofluorescence staining (DAPI and CD31) of TA muscle sections from WT and Hmgb1 +/− mice at day 5 after injury. Bars, 50 µm. (B) Quantification of CD31-positive cells/mm 2 in TA muscles of WT and Hmgb1 +/− mice at day 5 after injury. n = 4 mice per group of three independent experiments. (C) Quantitative PCR analysis of mRNA levels of angiopoietin-1 (Ang-1) in triceps at day 5 after injury. n = 6 mice per group of two independent experiments. (D) Representative immunostaining (DAPI and CD45, top; DAPI and CD68, bottom) of TA muscle sections from WT and Hmgb1 +/− mice at day 5 after injury. Bars, 50 µm. (E) Quantification of CD45-positive cells isolated with immunobeads from injured muscles. n = 6 mice per group of two independent experiments. (F) Quantification of CD68-positive cells ( n = 4 mice) and CD68/CD163-positive macrophages ( n = 3 mice) in TA muscle sections from WT and Hmgb1 +/− mice at day 5 after injury, two independent experiments. (G) Representative immunofluorescence staining for DAPI, laminin, and Pax7 in TA muscles from WT and Hmgb1 +/− mice at day 5 after injury (Pax7-positive cells indicated with white arrowheads). Bars, 50 µm. (H) Quantification of Pax7-positive cells in TA muscles of WT and Hmgb1 +/− mice at day 5 after injury. n = 3 mice per group of two independent experiments. In all panels, data are means ± SEM, and statistical significance was assessed with Student’s t test. *, P < 0.05; **, P < 0.01.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Expressing, Injection, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Immunostaining, Isolation

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: 3S and BoxA directly bind to CXCR4. (A and B) SPR sensorgrams representing the binding of HMGB1 isoforms to immobilized MD-2 (A) or sRAGE (B). ds-HMGB1 and 3S bind to MD-2 with an apparent K D of 0.35 µM and 2 µM, respectively (A). Binding to sRAGE: apparent K D of 1 µM for ds-HMGB1 and 3.5 µM for fr-HMGB1 and 3S (B). Data are representative of three independent experiments. (C) TA muscles of WT, Tlr4 −/− and Rage −/− mice were injected with Ctx and vehicle (PBS) or 3S, and the CSA of centronucleated fibers was measured at day 5 after injury. n = 3 mice per group, two independent experiments. Data are means ± SEM. Student t tests within each genotype: *, P < 0.05; **, P < 0.01. (D) Sensorgrams representing the binding of 40 nM fr-HMGB1 or 3S, 50 nM CXCL12, and 40 nM BoxA to immobilized lentiviral particles with membrane-bound CXCR4. (E) Sensorgrams representing the binding of different concentrations of 3S (left) or BoxA (right) to immobilized lentiviral particles with membrane-bound CXCR4. Curves derived from these assays were analyzed by fitting to a simple one-site interaction model with BIA Evaluation 4.1 software (GE Healthcare). Data are representative of three independent experiments. (F) Migration of WT ( Cxcr4 +/+ ) or Cxcr4 −/− MEFs toward 0.4 nM fr-HMGB1 or 3S. Data are representative of three independent experiments and are means ± SEM. Statistical significance between genotypes was calculated with Student’s t test. ***, P < 0.001; ****, P < 0.0001. (G and H) Migration of myoblasts toward 40 nM fr-HMGB1 and 3S with or without 10 nM anti-CXCL12 antibody (G) or toward 40 nM 3S and 30 nM CXCL12 with or without 250 nM BoxA (H). Data are means ± SEM of triplicates, two independent experiments. Differences between treatments were assessed with one-way ANOVA plus Tukey’s post-test. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Binding Assay, Injection, Derivative Assay, Software, Migration

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: HMGB1 supports muscle regeneration via CXCR4. (A–E) Comparison of C57BL/6 mice treated with Ctx plus vehicle versus Ctx plus BoxA and Ctx plus 5 mg/kg 3S with or without 10 mg/kg BoxA. (A) Laminin and DAPI staining of TA muscle sections at day 5 after injury. Bar, 50 µm. Number (B) and CSA of centronucleated fibers (CNFs; C) in TA muscles at day 5 after injury. n = 4 mice per group, two independent experiments. (D) Quantification of Pax7 + cells in regenerating TA muscles at day 5 after injury. n = 3 mice per group, two independent experiments. (E) Quantification of CD68 + CD163 + cells in sections of TA muscles at day 5 after injury. n = 4 mice per group, two independent experiments. (F–H) Comparison of muscles from mice receiving one single intramuscular injection of Ctx plus vehicle (PBS) versus Ctx plus 5 mg/kg AMD3100 with or without 5 mg/kg 3S. (F) Representative images of H&E and immunofluorescence staining for DAPI and laminin or CD68/CD163. Bars, 50 µm. (G) Number of CNFs in TA muscles at day 5 after injury. n = 3 mice per group, two independent experiments. (H) Quantification of CD68 + CD163 + cells on sections of TA muscles at day 5 after injury. n = 3 mice per group, two independent experiments. Differences between groups were assessed with one-way ANOVA plus Tukey’s post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Staining, Injection, Immunofluorescence

Journal: The Journal of Experimental Medicine

Article Title: High mobility group box 1 orchestrates tissue regeneration via CXCR4

doi: 10.1084/jem.20160217

Figure Lengend Snippet: 3S accelerates liver regeneration via CXCR4 . DILI was induced by i.p. injection of 300 mg/kg (body weight) APAP. 2 h later, mice received one single i.p. injection of vehicle (NaCl), 500 µg/mouse 3S, or 200 µg/mouse AMD3100 with or without 500 µg/mouse 3S. Serum collection and necroscopy were performed at different time points, and hepatic regeneration was assessed by i.p. injection of 1 mg/mouse BrdU 16 h before liver collection. (A) Representative images of H&E staining and HMGB1 immunostaining of liver in control mice on day 1 after DILI. HMGB1 negative nuclei (white arrowheads) are visible at higher magnification of the areas identified by rectangles in APAP-treated mice. Bars, 50 µm. (B) sALT and sAST in serum after APAP injection in mice treated with vehicle or 3S. n = 6 mice per group, two independent experiments. (C) Quantification of intrahepatic leukocytes (IHL) at days 1, 2, 3, 5, and 7 after APAP injection in control mice and after DILI in mice treated with vehicle or 3S. n = 4 mice per group, two independent experiments. Data are means ± SEM, and differences between groups were assessed with two-way ANOVA plus Bonferroni post-tests. The interaction between treatment and time is not significant. (D and E) Representative images of Ki-67 immunostaining (D) and quantification of Ki-67–positive hepatocytes (E) in livers from mice treated with vehicle or 3S at days 1, 2, 3, 5, and 7 after APAP injection. Bars, 50 µm. n = 4 mice per group, two independent experiments. Data are means ± SEM, and differences between groups were assessed with two-way ANOVA plus Bonferroni post-tests. The interaction between treatment and time is significant (P = 0.0003). (F) sALT and sAST after APAP injection in mice treated with vehicle, 3S, or AMD3100 with or without 3S. n = 4 mice per group, two independent experiments. Differences between groups in B and F were assessed with one-way ANOVA plus Dunnett’s post-test. (G and H) Representative images of BrdU immunostaining (G; BrdU-positive hepatocytes indicated with white arrowheads) and quantification of BrdU-positive hepatocytes (H) in livers from mice treated with vehicle, 3S, or AMD3100 with or without 3S at day 2 after APAP injection. Bars, 50 µm. n = 4 mice per group. Data are means ± SEM, and differences between groups were assessed with one-way ANOVA plus Bonferroni post-tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Livers were collected, fixed in zinc-formalin, and then embedded in paraffin and stained with H&E, anti–Ki-67 (1:100, clone SP6; Neomarkers) and anti-HMGB1 (1:200; BD PharMingen) and anti-BrdU (1:100, Abcam) antibodies.

Techniques: Injection, Staining, Immunostaining

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: The expression of F2/Rho, HMGB1/RAGE/p38MAPK/NF-κB signal pathway-related proteins increases in the lung tissues of rats simulated high-altitude hypoxia. (A) Western blot analysis was performed to examined the protein expression levels of F2, ROCK1, RhoA, HMGB1, RAGE, p38 MAPK and (NF-κB) p65 in the rat lung tissue. (B) Relative protein expression was normalized to that of the respective control, β-actin. (C-E) Immunohistochemistry of RAGE and HMGB1 in lung tissues of rats. The staining results of the images revealed that the expression of RAGE and HMGB1 in the lung tissues of rats exposed to hypoxia was significantly increased compared with that of the control rats. The left and right dark and bright images in each group is the comparison of the results before and after molecular staining. The positive results in the bright pictures refer to the immunostaining of HMGB1 and RAGE. * P<0.05 vs. the control group. F2, prothrombin; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1; C, control; H, hypoxia.

Article Snippet: The primary antibodies [HMGB1 (cat. no. R22773, Zen-Bio, Inc.; 1:1,000), RAGE (cat. no. ab216329, Abcam (1:1,000:)] were then added followed by incubation in a wet box at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Immunohistochemistry, Staining, Comparison, Immunostaining

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: Results of immunofluorescence staining of HMGB1/RAGE and ELISA of the inflammatory factors, TNF-α, IL-1β and IL-6, in the two groups of NR8383 cells. (A) From 0 to 24 h, cells in both the control and the anoxic group exhibited an increasing trend in viability; from 24 to 48 h, the cells in the control and the model group continued to exhibit cell growth, although the growth of the cells in the anoxic group was slower, and using an image cell analyzer, the cells at 24 h were more stable than those at the other time points. Therefore, the final anoxic conditions of NR8383 cells were 1% oxygen, 5% carbon dioxide and 94% nitrogen for 24 h. (B) The fluorescence intensity of HMGB1 protein in the control and hypoxia group. (C) The fluorescence intensity of RAGE protein in the control and hypoxia group. HMGB1 staining is red, DAPI staining is blue, RAGE staining is green. (D) The mean fluorescence intensity of RAGE and HMGB1 is presented. (E-G) ELISA was used to assess the contents of (E) TNF-α, (F) IL-1β and (G) IL-6 in the control and hypoxia group (24 h of culture). Magnification, ×40 and scale bar, 200 µ m. Data are presented as the mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001, vs. the control group. C, control; H, hypoxia; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1; TNF-α, tumor necrosis factor α; IL, interleukin.

Article Snippet: The primary antibodies [HMGB1 (cat. no. R22773, Zen-Bio, Inc.; 1:1,000), RAGE (cat. no. ab216329, Abcam (1:1,000:)] were then added followed by incubation in a wet box at 4°C overnight.

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Control, Fluorescence, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: The expression of F2/Rho, HMGB1/RAGE/p38MAPK/NF-κB signaling pathway-related proteins increased in the NR8383 cells with exposed to hypoxia. (A) Western blot analysis was performed to examine the protein expression levels of F2, ROCK1, RhoA, HMGB1, RAGE, p38MAPK and (NF-κB) p65. (B-H) Relative protein expression was normalized to that of the respective control, β-actin. Protein expression exhibited variable changes; * P<0.05, vs. the control group. C, control; H, hypoxia; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1.

Article Snippet: The primary antibodies [HMGB1 (cat. no. R22773, Zen-Bio, Inc.; 1:1,000), RAGE (cat. no. ab216329, Abcam (1:1,000:)] were then added followed by incubation in a wet box at 4°C overnight.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: Immunoprecipitation of F2 and RAGE reveals a direct interaction between the two proteins. In whole cell lysate group, F2 and RAGE were expressed at the molecular weight of normal protein (F2, 72 kDa; in the immunoprecipitation group, F2 and RAGE were co-expressed at the same location (~55 kDa). Comparing the expression of F2 and RAGE proteins in the two groups, it was found that there was a direct interaction between the two proteins. IP, immunoprecipitation; WCL, whole cell lysate; C, control; H, hypoxia; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1; F2, prothrombin.

Article Snippet: The primary antibodies [HMGB1 (cat. no. R22773, Zen-Bio, Inc.; 1:1,000), RAGE (cat. no. ab216329, Abcam (1:1,000:)] were then added followed by incubation in a wet box at 4°C overnight.

Techniques: Immunoprecipitation, Molecular Weight, Expressing, Control

Journal: International Journal of Molecular Medicine

Article Title: Inflammation and coagulation abnormalities via the activation of the HMGB1-RAGE/NF-κB and F2/Rho pathways in lung injury induced by acute hypoxia

doi: 10.3892/ijmm.2023.5270

Figure Lengend Snippet: The cells were randomly divided into four groups as follows: The control group, control + siRAGE group, hypoxia + siRAGE group and hypoxia group, and cultured according to the culture conditions (the concentration of siRAGE was 100 nM). (A) Western blot analysis was performed to examine the protein expression levels of RAGE in the different groups. (B) The relative protein expression gray scale statistics of RAGE. (C) Western blot analysis was performed to examine the protein expression levels of RAGE, HMGB1, p-p38, p-p65, HIF-1α and β-actin in NR8383 cells in the control, control + siRAGE, hypoxia + siRAGE and hypoxia groups. (D) The relative protein expression was normalized to that of the respective control, β-actin. Data are presented as the mean ± standard deviation. *** P<0.001 and **** P<0.0001, comparison between control group and hypoxia group; ### P<0.001 and #### P<0.0001, comparison between the hypoxia group and hypoxia + siRAGE group; ns, not significant; RAGE, receptor for advanced glycation end products; HMGB1, high mobility group protein-1.

Article Snippet: The primary antibodies [HMGB1 (cat. no. R22773, Zen-Bio, Inc.; 1:1,000), RAGE (cat. no. ab216329, Abcam (1:1,000:)] were then added followed by incubation in a wet box at 4°C overnight.

Techniques: Control, Cell Culture, Concentration Assay, Western Blot, Expressing, Standard Deviation, Comparison