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: Frontiers in Pharmacology

Article Title: Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia

doi: 10.3389/fphar.2024.1469223

Figure Lengend Snippet: Contains a summary of the Primer Sequence List.

Article Snippet: The following primary antibodies were used: rabbit anti-HMGB1 antibody (20 μg, CUSABIO), rabbit anti-AQP1 antibody (20 μL, BOSTER BIOLOGICAL TECHNOLOGY), rabbit anti-RAGE antibody (10 μL, MedChemExpress), rabbit anti-GAPDH antibody (100 μL, BOSTER) and mouse anti-TLR4 antibody (20 μL, proteintech).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia

doi: 10.3389/fphar.2024.1469223

Figure Lengend Snippet: Design and characterization of HMGB1-interfering lentiviruses (A) Schematic design of shRNA targeting HMGB1 mRNA. (B) Real-time PCR analysis of HMGB1 RNA levels in rat astrocytes infected with lentivirus expressing HMGB1 shRNA. (C) qRT-PCR analysis of HMGB1 gene expression in the spinal cord of rats injected with different titers. **** p < 0.0001 *** p < 0.001 ** p < 0.01* p < 0.05.

Article Snippet: The following primary antibodies were used: rabbit anti-HMGB1 antibody (20 μg, CUSABIO), rabbit anti-AQP1 antibody (20 μL, BOSTER BIOLOGICAL TECHNOLOGY), rabbit anti-RAGE antibody (10 μL, MedChemExpress), rabbit anti-GAPDH antibody (100 μL, BOSTER) and mouse anti-TLR4 antibody (20 μL, proteintech).

Techniques: shRNA, Real-time Polymerase Chain Reaction, Infection, Expressing, Quantitative RT-PCR, Gene Expression, Injection

Journal: Frontiers in Pharmacology

Article Title: Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia

doi: 10.3389/fphar.2024.1469223

Figure Lengend Snippet: Mechanical and thermal pain threshold responses in CCD rats within each group (A) Mechanical pain thresholds in CCD rats after HMGB1 knockdown. (B) Thermal pain thresholds in CCD rats after HMGB1 knockdown. (C) Mechanical pain thresholds in CCD rats after TLR4 inhibition. (D) Thermal pain thresholds in CCD rats after TLR4 inhibition. (E) Mechanical pain thresholds in CCD rats after RAGE inhibition. (F) Thermal pain thresholds in CCD rats after RAGE inhibition. N = 5 per group **** p < 0.0001 *** p < 0.001 ** p < 0.01* p < 0.05 vs. CCD.

Article Snippet: The following primary antibodies were used: rabbit anti-HMGB1 antibody (20 μg, CUSABIO), rabbit anti-AQP1 antibody (20 μL, BOSTER BIOLOGICAL TECHNOLOGY), rabbit anti-RAGE antibody (10 μL, MedChemExpress), rabbit anti-GAPDH antibody (100 μL, BOSTER) and mouse anti-TLR4 antibody (20 μL, proteintech).

Techniques: Knockdown, Inhibition

Journal: Frontiers in Pharmacology

Article Title: Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia

doi: 10.3389/fphar.2024.1469223

Figure Lengend Snippet: Immunoprecipitation determinations of HMGB1 and AQP1 (A) Western blots of HMGB1 in plasmids. (B) Western blots of AQP1 in plasmids. (C) CO-IP assay results. 293T: 293T-null cells; 293T-E5061-E5077: 293T- E5061 HA empty control plasmid transfection- E5077 negative control CON238 plasmid; 293T-E5062-E5078: 293T- E5062 HA-Aqp1 overexpression plasmid transfection- E5078 Hmgb1-3flag overexpression plasmid. Flag: HMGB1; HA:AQP1.

Article Snippet: The following primary antibodies were used: rabbit anti-HMGB1 antibody (20 μg, CUSABIO), rabbit anti-AQP1 antibody (20 μL, BOSTER BIOLOGICAL TECHNOLOGY), rabbit anti-RAGE antibody (10 μL, MedChemExpress), rabbit anti-GAPDH antibody (100 μL, BOSTER) and mouse anti-TLR4 antibody (20 μL, proteintech).

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Control, Plasmid Preparation, Transfection, Negative Control, Over Expression

Journal: Frontiers in Pharmacology

Article Title: Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia

doi: 10.3389/fphar.2024.1469223

Figure Lengend Snippet: Changes in AQP1 expression after HMGB1 knockdown (A) Protein expression levels of HMGB1 and AQP1 in the spinal cords of rats in each group. (B) Protein expression levels of HMGB1 and AQP1 in the LPS inflammatory cell model in each group. (C) mRNA levels of HMGB1 and AQP1 in the spinal cords of rats in each group. (D) mRNA levels of HMGB1 and AQP1 in the LPS inflammatory cell model in each group. N = 3 per group **** p < 0.0001 *** p < 0.001 ** p < 0.01* p < 0.05.

Article Snippet: The following primary antibodies were used: rabbit anti-HMGB1 antibody (20 μg, CUSABIO), rabbit anti-AQP1 antibody (20 μL, BOSTER BIOLOGICAL TECHNOLOGY), rabbit anti-RAGE antibody (10 μL, MedChemExpress), rabbit anti-GAPDH antibody (100 μL, BOSTER) and mouse anti-TLR4 antibody (20 μL, proteintech).

Techniques: Expressing, Knockdown

Journal: Frontiers in Pharmacology

Article Title: Lentivirus-mediated RNA interference targeting HMGB1 modulates AQP1 to reduce pain induced by chronic compression of the dorsal root ganglia

doi: 10.3389/fphar.2024.1469223

Figure Lengend Snippet: Changes in NF-κB expression following knockdown of HMGB1 or AQP1 (A) Protein expression levels of HMGB1 and NF-κB in the spinal cords of rats in each group. (B) mRNA levels of HMGB1 and NF-κB in the spinal cords of rats in each group. (C) Protein expression levels of AQP1 and NF-κB in the spinal cords of rats in each group. (D) mRNA levels of AQP1 and NF-κB in the spinal cords of rats in each group. (E) Protein expression levels of NF-κB and AQP1 in the LPS inflammatory cell model in each group. (F) mRNA levels of NF-κB and AQP1 in the LPS inflammatory cell model in each group. N = 3 per group **** p < 0.0001 *** p < 0.001 ** p < 0.01* p < 0.05.

Article Snippet: The following primary antibodies were used: rabbit anti-HMGB1 antibody (20 μg, CUSABIO), rabbit anti-AQP1 antibody (20 μL, BOSTER BIOLOGICAL TECHNOLOGY), rabbit anti-RAGE antibody (10 μL, MedChemExpress), rabbit anti-GAPDH antibody (100 μL, BOSTER) and mouse anti-TLR4 antibody (20 μL, proteintech).

Techniques: Expressing, Knockdown

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: Ligand-activated PPARs inhibit LPS-induced release of HMGB1. ( a ) RAW 264.7 cells grown to 60% confluency were incubated in serum-free medium for 24 h, and then stimulated with LPS in the presence or absence of PPAR ligands for 24 h. ( b and c ) Cells were transfected with siRNA against PPAR- δ ( b ) or PPAR- γ ( c ), and grown for 38 h. After incubation in serum-free medium for 24 h, the cells were stimulated with LPS for 24 h in the presence or absence of GW501516 ( b ) or rosiglitazone ( c ). Equal volumes of conditioned media or aliquots of cell lysates were subjected to western blotting for determination of HMGB1 levels. An image analyzer was used to quantitate band intensity, and the ratios of HMGB1 to Ponceau S are shown. The results are expressed as means±S.E.M. ( n =3). ** P <0.01 compared with the untreated group; ## P <0.01 compared with the LPS-treated group; †† P <0.01 compared with the group treated with LPS+GW501516 or rosiglitazone. Ponceau S staining or β -actin was used as a loading control

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Incubation, Transfection, Western Blot, Staining, Control

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: Acetylation is involved in the regulation of HMGB1 release. ( a ) RAW 264.7 cells incubated in serum-free medium for 24 h were pretreated with resveratrol or sirtinol for 1 h, and then stimulated with LPS for 6 h (for detection of acetyl-HMGB1) or 24 h (for detection of HMGB1 released). ( b ) Cells transfected with SIRT1 siRNA or control siRNA were grown for 38 h, after which they were incubated in serum-free medium for 24 h, and then stimulated with LPS for 6 h (for detection of acetyl-HMGB1) or 24 h (for detection of released HMGB1). Cell lysates were pulled down with anti-HMGB1 and immunoblotted with anti-acetyl-lysine to detect acetylated HMGB1. Each membrane was then stripped and re-probed for total HMGB1, as a loading control. For determination of released HMGB1, equal volumes of conditioned media were subjected to western blot analysis; Ponceau S staining was used as a loading control. Whole-cell lysates were subjected to Western blot analysis to determine the expression levels of HMGB1 ( a ) and SIRT1 ( b ). An image analyzer was used to quantitate band intensity, and the fold changes in the acetyl-HMGB1 to HMGB1 or HMGB1 to Ponceau S ratio are shown. The results are expressed as means±S.E.M. ( n =3). ** P <0.01, * P <0.05 compared with the untreated group; ## P <0.01, # P <0.05 compared with the LPS-treated group. Ponceau S staining or β -actin was used as a loading control

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Incubation, Transfection, Control, Membrane, Western Blot, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: SIRT1 mediates the effects of PPAR- δ and - γ in the inhibition of LPS-primed release of HMGB1 through deacetylation. ( a ) RAW 264.7 cells grown to 60% confluency were incubated with serum-free medium for 24 h and then stimulated with LPS for the indicated times. ( b ) Cells incubated for 24 h in serum-free medium were stimulated with LPS for 6 h in the presence or absence of the ligand indicated. ( c and d ) Cells were transfected with SIRT1 siRNA and grown for 38 h, after which they were stimulated with LPS for 6 h in the presence or absence of GW501516 ( c ) or rosiglitazone ( d ). Whole-cell lysates were immunoprecipitated with anti-HMGB1, and then acetylated HMGB1 was detected by western blotting with an anti–acetyl-lysine antibody. An image analyzer was used to quantitate band intensity, and the ratios of acetylated HMGB1 to total HMGB1 are shown. The results are expressed as means±S.E.M. ( n =3). * P <0.05 compared with the untreated group; # P <0.05 compared with the LPS-treated group; † P <0.05 compared with the group treated with LPS+GW501516 or rosiglitazone

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Inhibition, Incubation, Transfection, Immunoprecipitation, Western Blot

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: SIRT1 is essential for the inhibition of LPS-induced HMGB1 release by PPAR- δ and - γ . ( a and b ) RAW 264.7 cells pretreated with resveratrol ( a ) or sirtinol ( b ) for 1 h were stimulated with LPS in the presence or absence of GW501516. ( c and d ) Cells pretreated with resveratrol ( c ) or sirtinol ( d ) for 1 h were incubated with LPS in the presence or absence of rosiglitazone. After incubation for 24 h, equal volumes of conditioned media were analyzed by western blotting with an anti-HMGB1 antibody. An image analyzer was used to quantitate band intensity, and the ratios of HMGB1 to Ponceau S are shown. The results are expressed as means±S.E.M. ( n =3). ** P <0.01 compared with the untreated group; ## P <0.01 compared with the LPS-treated group; †† P <0.01 compared with the group treated with LPS+GW501516 or rosiglitazone. Ponceau S staining was used as a loading control

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Inhibition, Incubation, Western Blot, Staining, Control

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: Downregulation of SIRT1 abrogates the prevention of PPAR- δ and - γ against HMGB1 release. ( a ) RAW 264.7 cells grown to 60% confluency were incubated with serum-free medium for 24 h, and then stimulated with LPS in the presence or absence of the indicated ligand for 24 h. Aliquots of protein from cell lysates were analyzed by western blotting with anti-SIRT1 or anti- β -actin antibody. ( b and c ) Cells transfected with siRNA against SIRT1 were stimulated with LPS in the presence or absence of the GW501516 ( b ) or rosiglitazone ( c ) for 24 h. Equal volumes of conditioned media were analyzed by western blotting with anti-HMGB1 antibody. An image analyzer was used to quantitate band intensity, and the fold changes in the HMGB1 to Ponceau S or SIRT1 to β -actin ratio are shown. The results are expressed as means±S.E.M. ( n =3). ** P <0.01 compared with the untreated group; ## P <0.01, # P <0.05 compared with the LPS-treated group; †† P <0.01 compared with the group treated with LPS+GW501516 or rosiglitazone. Ponceau S staining or β -actin was used as a loading control

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Incubation, Western Blot, Transfection, Staining, Control

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: SIRT1-mediated deacetylation of HMGB1 is a critical factor in the PPAR- δ / γ –mediated inhibition of HMGB1 release. ( a ) RAW 264.7 cells were transfected with empty vector (pcDNA3.1/Myc) or pcDNA3.1-Myc-SIRT1. ( b ) Cells were transfected with empty vector (pcDNA3.1/HA) or pcDNA3.1-HA-PCAF. ( c ) Cells were transfected with empty vector (pcDNA3.1), pcDNA3.1-Myc-SIRT1, or pcDNA3.1-HA-PCAF. After incubation for 38 h, cells were maintained in serum-free medium for 24 h, and then stimulated with or without LPS for 6 h (for detection of acetyl-HMGB1) or 24 h (for detection of released HMGB1). Cell lysates were pulled down with anti-HMGB1 and immunoblotted with anti-acetyl-lysine to detect acetylated HMGB1. Each membrane was then stripped and re-probed for HMGB1, as a loading control. For determination of released HMGB1, equal volumes of conditioned media were subjected to Western blot analysis; Ponceau S staining was used as a loading control. Whole-cell lysates were subjected to western blot analysis with an anti-HMGB1, anti-SIRT1, anti-Myc, or anti-HA antibody, as appropriate, to determine the expression levels of HMGB1 and SIRT1 ( a and c ) or PCAF ( b and c )

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Inhibition, Transfection, Plasmid Preparation, Incubation, Membrane, Control, Western Blot, Staining, Expressing

Journal: Cell Death & Disease

Article Title: Ligand-activated peroxisome proliferator-activated receptor- δ and - γ inhibit lipopolysaccharide-primed release of high mobility group box 1 through upregulation of SIRT1

doi: 10.1038/cddis.2014.406

Figure Lengend Snippet: Acetylated HMGB1 is an effective substrate for SIRT1. ( a ) HEK293T cells were transfected with empty vector (pcDNA3.1), pcDNA3.1-Flag-HMGB1, or pcDNA3.1-Myc-SIRT1. ( b ) HEK293T cells were transfected with pcDNA3.1-HA-PCAF or increasing amounts (2 μ g and 4 μ g) of pcDNA3.1-Myc-SIRT1. After incubation for 48 h, the cells were stimulated with ( a ) or without ( b ) LPS for 3 h. ( c ) Whole-cell lysates from SIRT1 -knockout ( SIRT1 −/− ) or wild-type ( SIRT1 +/+ ) MEFs were pulled down with anti-HMGB1 and analyzed by western blotting with anti-acetyl-lysine, anti-HMGB1, or anti-SIRT1 antibody to detect acetylated HMGB1 or total HMGB1 and SIRT1. ( d ) SIRT1 −/− MEFs were transfected with empty vector (pcDNA3.1-Myc) or pcDNA3.1-Myc-SIRT1 and incubated for 48 h, after which the cells were harvested and subjected to immunoprecipitation with anti-HMGB1 antibody. Acetylated HMGB1 or total HMGB1 and SIRT1 were detected by western blot analysis. β -actin was used as a loading control

Article Snippet: Immunoprecipitation was performed with 1 μ g of anti-HMGB1 antibody from 100 μ g whole-cell lysate protein in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).

Techniques: Transfection, Plasmid Preparation, Incubation, Knock-Out, Western Blot, Immunoprecipitation, Control