Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 decreases the expression of pro-inflammatory cytokines and catabolic mediators in rat synovium of CFA induced TMJOA. A) The schematic chart for the timeline of treating rats. B) The expression of ADAMTS5, MMP13, IL-1β, IL-6 and negative controls was detected by immunohistochemistry in the synovium of CFA groups, CFA with anti-HMGB1 antibody treatment groups at day 21, or in the control synovium; scale bar: 100 μm. C) Quantitative analysis of ADAMTS5, MMP13, IL-1β and IL- 6 in the synovium of rats; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, Expressing, Immunohistochemistry

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 alleviates the damage of cartilage and subchondral bone in CFA induced TMJOA. A) H&E, Safranin O and Masson trichrome staining of rat models at day 21; scale bar: 100 μm. B-D) Analysis of the condylar cartilage thickness, the Safranin O positive areas and unmineralized bone areas; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 decreases the expression of pro-inflammatory cytokines and catabolic mediators in rat cartilage of CFA induced TMJOA. A) The expression of ADAMTS5, MMP13, IL-1β, IL-6 and negative controls was detected by immunohistochemistry in the cartilage of CFA groups, CFA with anti-HMGB1 antibody treatment groups at day 21, or in the control cartilage; scale bar: 100 μm. B) Rate of ADAMTS5, MMP13, IL-1β and IL-6 positive cells in the cartilage was markedly increased in the CFA groups, while markedly inhibited after treatment with anti-HMGB1 antibody; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, Expressing, Immunohistochemistry

Journal: European Journal of Histochemistry : EJH

Article Title: Inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway

doi: 10.4081/ejh.2022.3357

Figure Lengend Snippet: Inhibition of HMGB1 suppresses the NF-κB signaling pathway in vivo. Expression of phospho-p65 in the synovium (A) and cartilage (B) of CFA groups, CFA with anti-HMGB1 antibody treatment groups at day 21 were detected by IF staining, or in the control groups; data are shown as the means with 95% CI and analyzed by one-way analysis of variance (ANOVA), n=5, *p<0.05, **p<0.01, ***p<0.001; scale bar: 100 μm. C) Schematic diagram of inhibition of HMGB1 suppresses inflammation and catabolism in temporomandibular joint osteoarthritis via NF-κB signaling pathway.

Article Snippet: After 2 weeks, 5 rats previously injected with CFA were intraperitoneally injected with 100 μg/kg/day anti HMGB1 antibody (MAB1690-100; R&D System) for 7 days.

Techniques: Inhibition, In Vivo, Expressing, Staining

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 2. CuB induced NLRP3/caspase-1/GSDMD-mediated pyroptosis. a The morphology changes of A549 cells after treatment with CuB (100 nM) for 8 h (Scale bar=25 µm). b The features of pyroptosis in A549 cells were detected by TEM (Scale bar=2 µm). c The expression of GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL-1β, and HMGB1 in A549 cells. d Immunofluorescence detection of the expression of HMGB1 in A549 cells (Scale bar=20 µm). e The EGFP-NLRP3 and OFPSpark-NEK7 fluorescence were examined by immunofluorescence assay (Scale bar=7.5 µm). f Co-IP analysis of the interaction between NEK7 and NLRP3 in A549 cells after treatment with CuB (100 nM) for 24 h in the presence of VX765 (60 µm). g The expression of NLRP3, NEK7, ASC, pro-caspase-1, and cleaved caspase-1 in A549 cells. h, i Immunofluorescence detection of the caspase-1 expression (Scale bar=25 µm) and ASC dot formation in A549 cells (Scale bar=7.5 µm). j Annexin V and 7-AAD double staining assay was used to confirm CuB (100 nM)-induced pyroptosis in A549 cells.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Immunofluorescence, Fluorescence, Co-Immunoprecipitation Assay, Double Staining

Journal: Pharmacological research

Article Title: Cucurbitacin B inhibits non-small cell lung cancer in vivo and in vitro by triggering TLR4/NLRP3/GSDMD-dependent pyroptosis.

doi: 10.1016/j.phrs.2021.105748

Figure Lengend Snippet: Fig. 7. CuB induced lung cancer cells pyroptosis in mouse models. a HE staining of heart, liver, spleen, lung, and kidney tissues in indicated groups (HE, original magnification, 200×, Scale bar=50 µm). b, c The mitochondrial ROS and cytosolic Ca2+ levels in tumor cells were detected by flow cytometry (n ≥3, ***P＜0.001 vs model group, ###P＜0.001 vs CuB 0.75 mg/kg group). d, e The protein expressions of TLR4, NEK7, NLRP3, ASC, Tom20, GSDMD-FL, GSDMD-N, pro-IL-1β, mature IL- 1β, pro-caspase-1, cleaved caspase-1and HMGB1 in tumor tissues were detected by western blotting.

Article Snippet: Antibodies for GSDMD (#93709), GAPDH (#8884), HMGB1 (#3935), cleaved-IL-1β (#83186), NLRP3 (#15101), cleaved-caspase1 (#4199), ASC (#13833), IL-1β (#12703), Gasdermin D (#93709), Tom20 (#42406), and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Staining, Flow Cytometry, Western Blot

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Changes in CCL5 and HMGB1 protein levels changes at lesion sites following rat SCI. (A) ELISA measurement of CCL5 protein levels at lesion sites following SCI at 0 d, 1 d, 4 d and 7 d, respectively. (B) Western blot analysis of HMGB1 expression following SCI at 0 d, 1 d, 4 d and 7 d. (C) Quantification data are shown in ( B ). Quantities were normalized to endogenous β-actin. (D) ELISA analysis of CCL5 protein levels at lesion sites at 0 d, 1 d, 4 d and 7 d following with or without intrathecal injection of 10 µl of an HMGB1 neutralizing antibody (HMGB1 Ab, 50 µg/kg). (E) Immunofluorescence staining revealed the colocalization of CCL5 with GFAP-positive astrocytes before or after SCI at 4 d with or without the intrathecal injection of 10 µl of HMGB1 Ab (50 µg/kg). The rectangle indicates the region magnified. Arrowheads indicate positive signals. n = 6. Scale bars, 50 μm in (E) . The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Injection, Immunofluorescence, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Examination of CCL5 production in astrocytes following stimulation with rHMGB1. ( A , B ) Purified primary astrocytes were stained with GFAP and Hoechst 33,342, and the purity was greater than 90%. Scale bar, 50 μm. ( C , D ) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following stimulation with 0–2.5 µg/mL rat recombinant HMGB1 (rHMGB1) for 24 h, respectively. ( E , F ) ELISA assay was used to determine the production of CCL5 in the lysates and supernatants following primary astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of 2.5 µg/ml HMGB1 Ab, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Purification, Staining, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of RAGE, TLR-2, or TLR-4 interference on HMGB1-induced CCL5 production in astrocytes. ( A , B ) Cell lysates and supernatants were tested by ELISA for the production of CCL5, following astrocyte treatment with 0.5 µg/ml rHMGB1 in the presence of the RAGE inhibitor FPS-ZM1 (2 µM) for 24 h. ( D , E ) ELISA was used to determine CCL5 protein levels in lysates and supernatants from astrocytes after stimulation with 0.5 µg/ml rHMGB1 in the presence of the TLR2 inhibitor C29 (10 µM) for 24 h. ( G , H ) CCL5 levels in lysates and supernatants of astrocytes were determined by ELISA after the astrocytes were treated with the TLR4 inhibitor TAK-242 (2 µM) in the presence of 0.5 µg/ml rHMGB1 for 24 h. ( C , F , I ) CCK8 assay of the effects of FPS-ZM1, C29, or TAK-242 on the viability of astrocytes. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of the inhibition of MAPK/NF-κB signaling on the HMGB1-induced production of CCL5 from astrocytes. ( A , B ) ELISA was used to determine the effects of 10 µM ERK inhibitor PD98059, 10 µM JNK inhibitor SP6001251 or 10 µM P38 inhibitor SB203580 in the presence of 0.5 µg/ml rHMGB1 for 24 h on CCL5 protein production levels in lysates and supernatants of astrocytes. (C) Western blot analysis of the activation of p65NFκB protein after astrocyte treatment with 10 µM ERK inhibitor PD98059 or 10 µM JNK inhibitor SP6001251 for 24 h in the presence of 0.5 µg/ml rHMGB1. (D) Quantification data are shown in (C). Quantities were normalized to endogenous β-actin. ( E , F ) ELISA analysis of CCL5 in the lysates and supernatants of primary astrocytes following challenge with 10 µM SN50 for 24 h in the presence of 0.5 µg/ml rHMGB1, respectively. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of HMGB1-mediated astrocytic CCL5 on the migration of BV2 microglia cells or RAW 264.7 macrophage cells in vitro. (A) Illustration of the BV2 or RAW 264.7 cells with rCCL5 coculture model. (B) Transwell assay analysis of migration the ability of BV2 or RAW 264.7 cells co-incubated with 0.1 µg/ml rCCL5 for 48 h. ( C ) and ( D ) Quantification data are shown in (B). (E) Illustration of the BV2 or RAW 264.7 cells and ACM coculture model. (F) Migration assay of BV2 or RAW 264.7 cells cocultured with ACM. ACM were prepared from astrocytes exposed to 0.5 µg/ml rHMGB1 for 24 h, followed by the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. (G) and (H) Quantification data are shown in (F). Scale bars, 100 μm in ( B ) and ( F ). The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Migration, In Vitro, Transwell Assay, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Effects of HMGB1-induced astrocytic CCL5 on microglia/macrophage polarization. ( A ) and ( B ) qRT-PCR was used to determine the expression of M1 markers ( CD86 , iNOS and TNF-a ) and M2 markers ( CD206 , Arg1 and Ym1 ) after BV2 or RAW 264.7 cells incubated with 0.1 µg/ml rCCL5 for 24 h. ( C ) and ( D ) The expression levels of M1 and M2 markers were determined by qRT-PCR following BV2 or RAW 264.7 cells cocultured with ACM for 24 h. ACM was prepared by astrocytes challenge with 0.5 µg/ml rHMGB1 for 24 h, after which the supernatant was collected and incubated with 2.5 µg/ml IgG or CCL5 Ab for 4 h. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Quantitative RT-PCR, Expressing, Incubation, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Immunofluorescence of microglia/macrophage and locomotor function assessment after the inhibition of HMGB1 or CCL5 by neutralizing antibodies in rat SCI. ( A ) Immunofluorescence of IBA1- or CD68-positive cells at 4 d following SCI after neutralizing antibody application by intrathecal injection of 10 µl of neutralizing antibodies (HMGB1 Ab, 50 µg/kg), CCL5 (CCL5 Ab, 50 µg/kg) or normal rabbit IgG (IgG, 50 µg/kg). n = 6. Scale bars, 50 μm. ( B , C ) Quantitative analysis of the intensity of IBA1- or CD68-labeled cells in ( A) , respectively. (D) HE staining of the injured spinal cord at 21 d after administration of 10 µl of HMGB1, CCL5 neutralizing antibodies or normal rabbit IgG. n = 6. Scale bars, 500 μm. (E) Quantification data are shown in ( D) . (F) Basso, Beattie, and Bresnahan (BBB) locomotor scale scores for hindlimb motor function in rats at 0 d, 7 d, 14 d, 21 d and 28 d following the intrathecal administration of the HMGB1 Ab, CCL5 Ab or normal rabbit IgG. n = 6. The experiments were performed in triplicate. The error bars represent the standard deviation (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Immunofluorescence, Inhibition, Injection, Labeling, Staining, Standard Deviation

Journal: Scientific Reports

Article Title: High mobility group box-1 protein promotes astrocytic CCL5 production through the MAPK/NF-κB pathway following spinal cord injury

doi: 10.1038/s41598-024-72947-2

Figure Lengend Snippet: Mechanistic diagram of HMGB1-mediated astrocytic CCL5 contributes to microglia/macrophages activation and recruitment.

Article Snippet: For drug delivery, 10 µl of HMGB1 (50 µg/kg, R&D Systems, cat. no. MAB16901), CCL5 neutralizing antibody (50 µg/kg, R&D Systems, cat. no. MAB478) or IgG control (50 µg/kg, R&D Systems, cat. no. 6-001-A) was then slowly injected (injection flow rate, 35 nl/s) intrathecally using the Hamilton syringe (needle size, 26 ga).

Techniques: Activation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release

doi: 10.3389/fcell.2024.1348707

Figure Lengend Snippet: List of proteins identified by tandem mass tag (TMT) proteomic analysis of A549 Lung cancer cells treated by oleic acid, arachidonic acid, or palmitic acid following overnight delipidation.

Article Snippet: Antibodies used in this study were the following: For immunoblotting, mouse monoclonal β-catenin (1:10,000; MilliporeSigma, SAB1305546), rabbit polyclonal HMGB1 (1:2,500; Novus, NB100-2322), mouse monoclonal PD-L1 (1:3,000; Sino Biologicals, 10084-MM33-50), rabbit monoclonal phospho-IRF3 (1:1,000, Cell Signaling 37829), rabbit polyclonal IRF3 (1:10,000, ProteinTech 11312-AP), rabbit monoclonal NF-kappa B p65 (1:1,000, Cell Signaling 8242), rabbit monoclonal phospho-NF-kappa B p65 (1:1,000, Cell Signaling, 3033).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release

doi: 10.3389/fcell.2024.1348707

Figure Lengend Snippet: Proteins involved in HMGB1 and MUFA synthetic pathways are inversely correlated in NSCLC. (A) Analysis of TCGA lung adenocarcinoma RNA-seq datasets from UCSC Xena project comparing HMGB1 gene expression in lung tumors vs. normal tissue using GEPIA webserver. (B) Kaplan-Meier plot of median overall survival (OS) in groups with either high or low expression of HMGB1. (C) Quartile overall survival in groups with either high or low HMGB1 gene expression. (D) Pearson correlation analysis between the expression of SCD1 and HMGB1 in lung cancer patients. (E) Heatmap generated from Clinical Proteomic Tumor Analysis Consortium lung adenocarcinoma dataset comparing the expression proteins in lipogenic (FASN, SREBF1, SREBF2, SCD, SCD5, PLIN2, and PLIN3) and HMGB1 (AGER and HMGB1) pathways.

Article Snippet: Antibodies used in this study were the following: For immunoblotting, mouse monoclonal β-catenin (1:10,000; MilliporeSigma, SAB1305546), rabbit polyclonal HMGB1 (1:2,500; Novus, NB100-2322), mouse monoclonal PD-L1 (1:3,000; Sino Biologicals, 10084-MM33-50), rabbit monoclonal phospho-IRF3 (1:1,000, Cell Signaling 37829), rabbit polyclonal IRF3 (1:10,000, ProteinTech 11312-AP), rabbit monoclonal NF-kappa B p65 (1:1,000, Cell Signaling 8242), rabbit monoclonal phospho-NF-kappa B p65 (1:1,000, Cell Signaling, 3033).

Techniques: RNA Sequencing Assay, Expressing, Generated

Journal: Frontiers in Cell and Developmental Biology

Article Title: Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release

doi: 10.3389/fcell.2024.1348707

Figure Lengend Snippet: SCD inhibition promotes release of HMGB1 from lung cancer cells. (A) Cell-titer Glo viability assay of cells treated for 24 h with 1 µM SCD1 inhibitor, A939572. (B) Real-time quantitative PCR analysis for HMGB1 and SCD1 mRNA in A549 cells treated 24 h with 1 µM SCD1 inhibitor. (C) Immunoblot analysis of HMGB1 expression in nuclear and cytoplasmic fraction of A549 cells treated 24 h with increasing concentration of SCD1 inhibitor. (D) HMGB1-specific enzyme-linked immunosorbent assay on extracellular media from A549 cells treated 24 h with increasing concentration of SCD1 inhibitor. Error bars represent the standard error of the mean (S.E.M.), an * indicates a p -value of < 0.05.

Article Snippet: Antibodies used in this study were the following: For immunoblotting, mouse monoclonal β-catenin (1:10,000; MilliporeSigma, SAB1305546), rabbit polyclonal HMGB1 (1:2,500; Novus, NB100-2322), mouse monoclonal PD-L1 (1:3,000; Sino Biologicals, 10084-MM33-50), rabbit monoclonal phospho-IRF3 (1:1,000, Cell Signaling 37829), rabbit polyclonal IRF3 (1:10,000, ProteinTech 11312-AP), rabbit monoclonal NF-kappa B p65 (1:1,000, Cell Signaling 8242), rabbit monoclonal phospho-NF-kappa B p65 (1:1,000, Cell Signaling, 3033).

Techniques: Inhibition, Viability Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release

doi: 10.3389/fcell.2024.1348707

Figure Lengend Snippet: MUFA increases retention of HMGB1 in lung cancer cells in a SIRT-dependent manner. (A) Immunoblot protein analysis of multiple lung cancer cells lines treated with delipidation media (delipidated serum and 1 µM SCD1 inhibitor) and 4-h replenishment with oleate-BSA. (B) HMGB1-specific ELISA on extracellular media from A549 and HCC827 lung cancer cells following 16-h delipidation and 4-h oleate-BSA replenishment. (C) Fluorescence microscopy of HMGB1-GFP transfected A549 cells following 16-h delipidation and 4-h oleate-BSA replenishment and neutral lipid staining with Nile Red, along with quantitation of localization of HMGB1-GFP sig nals. (D) Immunoblot protein analysis of HMGB1-GFP transfected A549 cells from (C) . (E) Immunoblot protein analysis of A549 lung cancer cells treated SIRT1 inhibitor cambinol for 24-h. (F) HMGB1-specific ELISA on extracellular media from cells treated in (E) . (G) Immunoblot protein analysis of A549 cells treated with combination of cambinol and SCD1 inhibitor in the presence and oleate-BSA. (H) HMGB1-specific ELISA on extracellular media obtained from cells treated with SIRT1 and SCD1 inhibitors in (G) . (I) Immunoblot protein analysis of delipidated A549 cells replenished with oleic acid-BSA, 72 h after transfection with SIRT1-specific siRNA. Error bars represent the standard error of the mean (S.E.M.), an *, indicates a p -value < 0.05.

Article Snippet: Antibodies used in this study were the following: For immunoblotting, mouse monoclonal β-catenin (1:10,000; MilliporeSigma, SAB1305546), rabbit polyclonal HMGB1 (1:2,500; Novus, NB100-2322), mouse monoclonal PD-L1 (1:3,000; Sino Biologicals, 10084-MM33-50), rabbit monoclonal phospho-IRF3 (1:1,000, Cell Signaling 37829), rabbit polyclonal IRF3 (1:10,000, ProteinTech 11312-AP), rabbit monoclonal NF-kappa B p65 (1:1,000, Cell Signaling 8242), rabbit monoclonal phospho-NF-kappa B p65 (1:1,000, Cell Signaling, 3033).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, Microscopy, Transfection, Staining, Quantitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release

doi: 10.3389/fcell.2024.1348707

Figure Lengend Snippet: Modulation of HMGB1 diminishes impact of A549-cond . Media on monocytes . (A) THP-1 NF-kB-SEAP reporter cell assay following 24-h incubation with conditioned media from lung cancer cells treated with increasing concentrations of recombinant HMGB1 protein. (B) THP-1 IRF3 and NF-kB reporter cell assay following exposure to conditioned media from A549 lung cancer cells treated with glycyrrhizin. (C) Immunoblot protein analysis of ERK, NF-kB, p38 signaling pathways in THP-1 cells treated with glycyrrhizin in the presence or absence of MUFA. (D) Immunoblot protein analysis of delipidated A549 cells replenished with BSA, oleic acid-BSA, or palmitic acid-BSA; 72 h after transfection with HMGB1-specific siRNA. (E) HMGB1-specific ELISA on extracellular media from cells described in . (F) THP-1 IRF3-Luc and NF-kB-SEAP reporter assays following 24-h incubation of reporter cells with conditioned media from A549 cells described in . Error bars represent the standard error of the mean (S.E.M.), an *, indicates a p -value < 0.05 when comparing the sample to BSA control; a #, indicates a p -value < 0.05 when comparing the sample to the control siRNA counterpart.

Article Snippet: Antibodies used in this study were the following: For immunoblotting, mouse monoclonal β-catenin (1:10,000; MilliporeSigma, SAB1305546), rabbit polyclonal HMGB1 (1:2,500; Novus, NB100-2322), mouse monoclonal PD-L1 (1:3,000; Sino Biologicals, 10084-MM33-50), rabbit monoclonal phospho-IRF3 (1:1,000, Cell Signaling 37829), rabbit polyclonal IRF3 (1:10,000, ProteinTech 11312-AP), rabbit monoclonal NF-kappa B p65 (1:1,000, Cell Signaling 8242), rabbit monoclonal phospho-NF-kappa B p65 (1:1,000, Cell Signaling, 3033).

Techniques: Incubation, Recombinant, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Oleate alters the immune response in non-small cell lung adenocarcinoma through regulation of HMGB1 release

doi: 10.3389/fcell.2024.1348707

Figure Lengend Snippet: MUFA increases cell-associated HMGB1 and decreases PD-L1 in NSCLC and monocytes . (A) Immunoblot protein analysis of HMGB1 and PD-L1 expression in A549 cells treated for 4 h with increasing concentrations of oleate-BSA. (B) Immunoblot protein analysis of PD-L1 expression in THP-1 monocytes following 24-h incubation in presence or absence of delipidated A549 conditioned media. (C) Heatmap of multi-omic data from lung adenocarcinoma published dataset examining expression of HMGB1, SCD1, SREBF1, and PD-L1 (CD274) along with several lipogenic genes and top hits from our proteomic analysis from . (D) Spearman’s correlation analysis among the select proteins from (C) and body mass index in female and male lung adenocarcinoma patients. (E) Pearson correlative analysis between expression of HMGB1 protein and PD-L1 (CD274) in female and male lung adenocarcinoma patients. (F) HMGB1-specific ELISA on serum samples from lung adenocarcinoma patients (n = 5), compared to histological detection of tumor-associated PD-L1. Any signal was considered positive for PD-L1 expression using the clinically validated Ventana assay. (G) Proposed mechanism of MUFA-mediated suppression of HMGB1 immune modulation in the tumor microenvironment. Error bars represent the standard error of the mean (S.E.M.), an *, indicates a p -value < 0.05 when comparing the sample to control.

Article Snippet: Antibodies used in this study were the following: For immunoblotting, mouse monoclonal β-catenin (1:10,000; MilliporeSigma, SAB1305546), rabbit polyclonal HMGB1 (1:2,500; Novus, NB100-2322), mouse monoclonal PD-L1 (1:3,000; Sino Biologicals, 10084-MM33-50), rabbit monoclonal phospho-IRF3 (1:1,000, Cell Signaling 37829), rabbit polyclonal IRF3 (1:10,000, ProteinTech 11312-AP), rabbit monoclonal NF-kappa B p65 (1:1,000, Cell Signaling 8242), rabbit monoclonal phospho-NF-kappa B p65 (1:1,000, Cell Signaling, 3033).

Techniques: Western Blot, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS medicine

Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

doi: 10.1371/journal.pmed.1000010

Figure Lengend Snippet: Figure 7. HMGB1 Is Secreted from Dying Tumor Cells and Is Required for Ad-Flt3L and Ad-TK Mediated Brain Tumor Regression (A) GL26 cells were infected with Ad-TK and incubated with 25 lM GCV. A western blot was performed using an antibody specific for HMGB1. The graph displays a quantification of the total amount of HMGB1 in the media of cells 24 h (yellow bars), 48 h (orange bars), or 72 h (black bars) after GCV treatment. (B) GL26 cells were incubated with Ad-TK (with and without GCV and glycyrrhizin), and media was overlaid on HEK293 reporter cells transfected with either a plasmid encoding TLR2 (þpTLR2; black) or with a control plasmid (-pTLR2; red). NFjB activity was determined by quantifying the activity of Firefly Luciferase (under the control of the NFjB promoter), *, p , 0.05 (Kruskal-Wallis followed by Dunn’s test). Inset: cells were incubated with 100 ng/ ml PAM3 (PAM3CSK4) as a positive control. (C) The levels of HMGB1 in mouse serum were quantified by ELISA 7 d after treatment of brain tumors with Ad-Flt3L and Ad-TK. *, p , 0.05 versus saline (Mann-Whitney U-test). (D) GL26 cells were implanted into C57BL/6 mice (n ¼ 5 mice/treatment group) and 17 d later were treated with saline, or with AdFlt3L and Ad-TK (F/T). Glycyrrhizin (Glyc), HMGB1-depleting (aHMGB1), or rabbit IgG isotype control antibodies were administered IP 2 d, 5 d, and 10 d after treatment, *, p , 0.05 Ad-Flt3L and Ad-TK versus Ad-Flt3L and Ad-TK þ glycyrrhizin; ^p , 0.05 Ad-Flt3L and Ad-TK þ isotype versus Ad-Flt3L and Ad-TK þ aHMGB1; Mantel log-rank test. doi:10.1371/journal.pmed.1000010.g007

Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

Techniques: Infection, Incubation, Western Blot, Transfection, Plasmid Preparation, Control, Activity Assay, Luciferase, Positive Control, Enzyme-linked Immunosorbent Assay, Saline, MANN-WHITNEY

Journal: PLoS medicine

Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

doi: 10.1371/journal.pmed.1000010

Figure Lengend Snippet: Figure 8. Western Blot Analysis Reveals HMGB1 Is Released from GL26, LLc1, GL261, and B16-F10 Cell Lines Following Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (TMZ) (A) LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0 at an MOI¼500. Noninfected cells were used as control. Ad-TK treated cells were also incubated with 25 lM GCV. 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (B) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as control. 72 h later, supernatant was collected and HMGB1 release was assessed by western blotting. (C) GL26, LLc1, GL261, or B16-F10 cells were incubated with temozolomide (TMZ) (200 lM). Control cells were incubated without TMZ (mock). 48 h later, supernatant was collected and HMGB1 release was assessed by western blotting. doi:10.1371/journal.pmed.1000010.g008

Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

Techniques: Western Blot, Irradiation, Infection, Control, Incubation

Journal: PLoS medicine

Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

doi: 10.1371/journal.pmed.1000010

Figure Lengend Snippet: Figure 9. HMGB1 Is Released into the Supernatant of GL26, LLc1, GL261, and B16-F10 Tumor Cell Lines in Response to Treatment with Ad-TK (þGCV), Irradiation, or Temozolomide (A–D) GL26, LLc1, GL261, or B16-F10 cells were infected with Ad-TK or Ad-0. Noninfected cells were used as control (mock). Ad-TK treated cells were incubated with 25 lM GCV. 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with Ad-TK (þGCV) significantly increased HMGB1 release when compared to corresponding mock and Ad0 treatment groups from each cell line, *, p , 0.05 versus mock and Ad-0 (Kruskal-Wallis test followed by Dunn’s test). (E–H) GL26, LLc1, GL261, or B16-F10 cells were irradiated with 20 Gy for 30 min. Nonirradiated cells were used as controls (mock). 72 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with irradiation significantly increased HMGB1 release when compared to corresponding control groups from each cell line, *, p , 0.05 versus mock (Mann-Whitney U-test). (I–L) GL26, LLc1, GL261, or B16-F10 cells (2.53105 cells per flask) were incubated with or without temozolomide (TMZ) (200 lM). 48 h later, supernatant was collected, and HMGB1 release was assessed by ELISA. Treatment with TMZ significantly increased HMGB1 release when compared to control groups from each cell type. *, p , 0.05 versus mock (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g009

Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

Techniques: Irradiation, Infection, Control, Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: PLoS medicine

Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

doi: 10.1371/journal.pmed.1000010

Figure Lengend Snippet: Figure 10. Treatment of Animals Bearing Intracranial Glioma (GL261) or Melanoma (B16-F10) with Ad-Flt3L and Ad-TK (þGCV) Results in Long-Term Survival and Increased Serum Levels of HMGB1 (A) GL261 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L (n ¼ 5), Ad-TK (þGCV) (n ¼ 6), Ad-Flt3L plus Ad-TK (þGCV) (n ¼ 5), or saline (n ¼ 5). Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad-Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (B) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of GL261 tumor-bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). (C) B16-F10 cells were implanted into the striatum of C57/Bl6 mice. 17 d later animals were treated with Ad-Flt3L, Ad-TK (þGCV), Ad-Flt3L plus Ad-TK (þGCV), or saline. Treatment with Ad-Flt3L and Ad-TK significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test), Ad- Flt3L, or Ad-TK alone treatment groups (^, p , 0.05; Mantel log-rank test). Treatment with Ad-Flt3L, or Ad-TK alone, significantly improved survival when compared to saline (*, p , 0.05; Mantel log-rank test). (D) 7 d after treatment, tumor-bearing animals were humanely killed and HMGB1 release was assessed in the serum. Treatment of B16-F10 tumor- bearing animals with Ad-Flt3L and Ad-TK (þGCV) significantly increased the levels of HMGB1 in the serum when compared to tumor-bearing animals treated with saline. *, p , 0.05 versus saline (Mann-Whitney U-test). doi:10.1371/journal.pmed.1000010.g010

Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

Techniques: Saline, MANN-WHITNEY

Journal: PLoS medicine

Article Title: HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

doi: 10.1371/journal.pmed.1000010

Figure Lengend Snippet: Figure 11. Model Illustrating the Role of TLR2 and HMGB1 in Initiating T Cell-Dependent Brain Tumor Regression by Flt3L and TK Treatment of tumor cells with TK þ GCV releases endogenous TLR2 ligands including HMGB1. DC infiltration into tumors and subsequent activation require TLR2 signalling. DC phagocytose tumor antigen and migrate to the dLN where they stimulate T cell clonal expansion resulting in T cell- dependent brain tumor regression and long-term immunological memory. doi:10.1371/journal.pmed.1000010.g011

Article Snippet: Generating HMGB1-Depleting Antibodies Antibodies were custom generated by New England Peptides and raised in New Zealand White rabbits against the N-terminal 11 amino acids of HMGB1 (MGKGDPKKPRG) (see Figure S3) [39].

Techniques: Activation Assay