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: World Journal of Gastroenterology

Article Title: Abdominal paracentesis drainage ameliorates myocardial injury in severe experimental pancreatitis rats through suppressing oxidative stress

doi: 10.3748/wjg.v26.i1.35

Figure Lengend Snippet: Effects of abdominal paracentesis drainage on pancreatic histopathology and proinflammatory cytokines. A: Representative micrographs of hematoxylin-eosin stained sections of rat pancreatic tissue from different groups. Images were taken under 100 × and 200 × magnification. The arrow indicates necrotic pancreatic tissue; B: Histology severity score of pancreas; C: Amylase; D: Interleukine-1 beta; E: Tumor necrosis factor alpha; F: Endotoxin; G: High mobility group box 1. Data indicate the mean ± standard deviation obtained from six animals in each group (C-G). a P < 0.05 vs sham group; c P < 0.05 vs severe acute pancreatitis group. IL-1β: Interleukine-1 beta; TNF-α: Tumor necrosis factor alpha; HMGB1: High mobility group box 1; PAAF: Pancreatitis associated ascitic fluids; SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage.

Article Snippet: Nonimmune chicken IgY (Boster Biological Technology, Wuhan, China) acted as a control antibody for HMGB1 neutralization.

Techniques: Histopathology, Staining, Standard Deviation

Journal: World Journal of Gastroenterology

Article Title: Abdominal paracentesis drainage ameliorates myocardial injury in severe experimental pancreatitis rats through suppressing oxidative stress

doi: 10.3748/wjg.v26.i1.35

Figure Lengend Snippet: Effects of pancreatitis associated ascitic fluids intraperitoneal injection with or without anti-high mobility group box 1 neutralizing antibody on cardiac NOX expression in cerulein rats. A: High mobility group box 1 in the serum; B: Nicotinamide adenine dinucleotide phosphate oxidase-2 ( NOX2 ) and NOX4 mRNA measurement by real-time polymerase chain reaction; C: Immunoblot of NOX2 and NOX4 protein expression from heart samples; D: Densitometry analysis of NOX2 and NOX4. Data indicate the mean ± standard deviation obtained from six animals in each group (A-B). Data are representative of at least three independent experiments (C-D). a P < 0.05 vs controls group; c P < 0.05 vs pancreatitis-associated ascitic fluid injection group. HMGB1: High mobility group box 1; NOX2: Nicotinamide adenine dinucleotide phosphate oxidase-2; NOX4: Nicotinamide adenine dinucleotide phosphate oxidase-4; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; SAP: Severe acute pancreatitis; APD: Abdominal paracentesis drainage.

Article Snippet: Nonimmune chicken IgY (Boster Biological Technology, Wuhan, China) acted as a control antibody for HMGB1 neutralization.

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Journal: World Journal of Gastroenterology

Article Title: Abdominal paracentesis drainage ameliorates myocardial injury in severe experimental pancreatitis rats through suppressing oxidative stress

doi: 10.3748/wjg.v26.i1.35

Figure Lengend Snippet: Possible mechanisms responsible for the protective effects of abdominal paracentesis drainage on severe acute pancreatitis associated cardiac injury. During pancreatitis, high levels of high mobility group box 1 (HMGB1) in the bloodstream can trigger a lethal inflammatory process and participate in the development of remote organ injury. Following abdominal paracentesis drainage treatment, the levels of high mobility group box 1 in the circulation decrease significantly, resulting in inhibition of expression and activity of cardiac nicotinamide adenine dinucleotide phosphate oxidase, thus reactive oxygen species production markedly decreases. These events downregulate expression of caspase-associated proteins and alleviate apoptosis, thereby yielding beneficial effects. HMGB1: High mobility group box 1; APD: Abdominal paracentesis drainage; NOX: Nicotinamide adenine dinucleotide phosphate oxidase; ROS: Reactive oxygen species.

Article Snippet: Nonimmune chicken IgY (Boster Biological Technology, Wuhan, China) acted as a control antibody for HMGB1 neutralization.

Techniques: Inhibition, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Ginsenoside Rh2 Inhibits NLRP3 Inflammasome Activation and Improves Exosomes to Alleviate Hypoxia-Induced Myocardial Injury

doi: 10.3389/fimmu.2022.883946

Figure Lengend Snippet: Ginsenoside Rh2 inhibited NLRP3 inflammasome activation via HMGB1/NF-κB signaling to promote exosome therapy for myocardial injury. Representative images of western blotting (A, C) and quantitative data (B, D) of the protein levels of NLRP3 and HMGB1. One-way ANOVA test was used for statistical analyses. Bars represent means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 . ns, no significance.

Article Snippet: The membrane was blocked with 5% skim milk in TBST at room temperature and incubated with the primary antibodies, including CD63 (1:1,000, Abcam, USA), TSG101 (1:1,000, Abcam, USA), NF-κB p65 (1:100, Abcam, USA), HMGB1 (1:1,000, HuaBio, China), and NLRP3 (1:1,000, HuaBio, China) overnight at 4°C.

Techniques: Activation Assay, Western Blot