Journal: Scientific Reports

Article Title: HMGB1 promotes hair growth via the modulation of prostaglandin metabolism

doi: 10.1038/s41598-019-43242-2

Figure Lengend Snippet: HMGB1 stimulates hair shaft elongation in human hair follicles. Isolated human hair follicles were treated with 200 ng/ml HMGB1 or 1 μM minoxidil for 9 days. ( a ) The photo panels show representative hair follicles from each group at day 0 (left) and 9 (right). Scale bar = 1 mm ( b ) Quantification of hair shaft growth rates. Changes in hair shaft length were measured using a stereomicroscope at 3, 6, and 9 days after culturing the cells. Data are shown as the mean ± SD of five independent donors (at least 90 follicles in each experiment). p-values were determined by ANOVA followed by Bonferroni post-hoc test. # p < 0.05, comparison between HMGB1 vs. control; *p < 0.05, comparison between minoxidil vs. control. ( c ) The percentage of hair follicles in each cycle stage at 9 days after treatment with 200 ng/ml HMGB1 or 1 μM minoxidil was assessed by morphological analysis. Data are shown as mean ± SD. ( d ) Immunofluorescence staining for Ki-67 (green) was performed after incubation of hair follicles with 200 ng/ml HMGB1 or 1 μM minoxidil for 3 days. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. Scale bar = 50 μm. ( e ) Ki-67 + positive cells in the matrix region were quantified. The results are shown as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 compared with control group using one-way ANOVA.

Article Snippet: Depending on the experiment, the media were supplemented with different concentrations of recombinant human HMGB1 (HMGB1; R&D systems, Minneapolis, MN, USA) or HMGB1 with antibodies blocking receptor for advanced glycation end-products (RAGE-FC, R&D systems), anti-TLR2 (BioLegend, San Diego, CA, USA), or anti-TLR4 (eBiosicence, San Diago, CA, USA) neutralizing antibodies.

Techniques: Isolation, Comparison, Control, Immunofluorescence, Staining, Incubation

Journal: Scientific Reports

Article Title: HMGB1 promotes hair growth via the modulation of prostaglandin metabolism

doi: 10.1038/s41598-019-43242-2

Figure Lengend Snippet: HMGB1 increases prostaglandin E synthase transcription in human dermal papilla cells (hDPCs). ( a ) mRNA expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), cytosolic prostaglandin E (cPGES), aldo-keto reductase family 1 member C1 (AKR1C1), aldo-keto reductase family 1 member C3 (AKR1C3), carbonyl reductase-1 (CBR-1), prostaglandin D synthase (PTGDS), alkaline phosphatase (ALPL), and vascular endothelial growth factor (VEGF) was examined in hDPCs treated with 100 or 200 ng/ml HMGB1 by real-time PCR analysis. The relative mRNA levels were normalized to GAPDH . The results are expressed as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 compared to control group using ANOVA. ( b ) hDPCs were treated with 200 ng/ml HMGB1 for different incubation periods (0.5, 1, 2, and 4 h). The protein levels of COX-1, COX-2, mPGES-1, mPGES-2, and cPGES were measured using western blot analysis. β-actin served as a loading control. The results are expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group using one-way ANOVA followed by Bonferroni’s test. Representative images of immunofluorescence staining for ( c ) COX-1, ( d ) COX-2, and ( e ) mPGES-1 in hDPCs cultured with 200 ng/ml HMGB1 for 30 min. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. Data are representative of three independent experiments. Scale bar = 20 μm. ( f ) PGE 2 secretion from hDPCs was determined by ELISA following treatment with various concentrations of HMGB1 (50, 100, and 200 ng/ml) for 4 h. The results are expressed as mean ± SD of three independent experiments. *p < 0.05 compared with control group using one-way ANOVA.

Article Snippet: Depending on the experiment, the media were supplemented with different concentrations of recombinant human HMGB1 (HMGB1; R&D systems, Minneapolis, MN, USA) or HMGB1 with antibodies blocking receptor for advanced glycation end-products (RAGE-FC, R&D systems), anti-TLR2 (BioLegend, San Diego, CA, USA), or anti-TLR4 (eBiosicence, San Diago, CA, USA) neutralizing antibodies.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Incubation, Western Blot, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: HMGB1 promotes hair growth via the modulation of prostaglandin metabolism

doi: 10.1038/s41598-019-43242-2

Figure Lengend Snippet: HMGB1 increases prostaglandin E synthase expression in human hair follicles. ( a ) Hematoxylin and eosin staining on the lower part of a cultured hair follicle. DP, dermal papilla. ( b ) Immunofluorescence staining for COX-1 (red) and mPGES-1 (green) in human hair follicles after 3 days of treatment with 200 ng/ml HMGB1 or 1 μM Minoxidil. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. Data are representative of ~4–5 hair follicles per each condition from three independent experiments. Scale bar = 50 μm.

Article Snippet: Depending on the experiment, the media were supplemented with different concentrations of recombinant human HMGB1 (HMGB1; R&D systems, Minneapolis, MN, USA) or HMGB1 with antibodies blocking receptor for advanced glycation end-products (RAGE-FC, R&D systems), anti-TLR2 (BioLegend, San Diego, CA, USA), or anti-TLR4 (eBiosicence, San Diago, CA, USA) neutralizing antibodies.

Techniques: Expressing, Staining, Cell Culture, Immunofluorescence

Journal: Scientific Reports

Article Title: HMGB1 promotes hair growth via the modulation of prostaglandin metabolism

doi: 10.1038/s41598-019-43242-2

Figure Lengend Snippet: RAGE is the corresponding receptor for HMGB1 in hDPCs. Blocking RAGE (RAGE-FC) inhibited the effects of HMGB1 on prostaglandin metabolism. Cultured hDPCs were pre-treated with or without 10 μg/ml RAGE-FC for 30 min and then incubated with 200 ng/ml HMGB1 for 30 min. ( a ) The protein levels of COX-1, COX-2, mPGES-1, and mPGES-2 were measured using western blot analysis. ( b ) Protein bands were analysed using densitometry, and β-actin served as a loading control. The results are expressed as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group using one-way ANOVA followed by Bonferroni’s test. ( c ) PGE 2 production from cultured hDPCs was measured using ELISA. Cultured hDPCs were pre-treated with 10 μg/ml RAGE-FC for 30 min and then incubated with 200 ng/ml HMGB1 for 4 h. Data are representative of three independent experiments. The results are expressed as mean ± SD of three independent experiments. **p < 0.01 and ***p < 0.001 compared with control group using one-way ANOVA. Representative images of immunofluorescence staining for ( d ) COX-1 (red), ( e ) COX-2 (red), and mPGES-1 (green) in hDPCs pre-treated with 10 μg/ml RAGE-FC for 30 min and 200 ng/ml HMGB1 treatment for another 30 min. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. White arrowheads mark the expression of each enzyme in perinuclear region of hDPCs. Data are representative of three independent experiments. Scale bar = 20 μm.

Article Snippet: Depending on the experiment, the media were supplemented with different concentrations of recombinant human HMGB1 (HMGB1; R&D systems, Minneapolis, MN, USA) or HMGB1 with antibodies blocking receptor for advanced glycation end-products (RAGE-FC, R&D systems), anti-TLR2 (BioLegend, San Diego, CA, USA), or anti-TLR4 (eBiosicence, San Diago, CA, USA) neutralizing antibodies.

Techniques: Blocking Assay, Cell Culture, Incubation, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: HMGB1 promotes hair growth via the modulation of prostaglandin metabolism

doi: 10.1038/s41598-019-43242-2

Figure Lengend Snippet: RAGE blockade inhibits HMGB1-induced hair shaft elongation in human hair follicles. Human hair follicles were pre-treated with 10 μg/ml RAGE-FC for 1 h followed by 200 ng/ml HMGB1 for 9 days. ( a ) Representative photos of hair follicles from each group are shown. ( b ) Quantification of hair shaft growth rates. The changes in hair shaft lengths were quantified at 6 and 9 days using a stereomicroscope. Data are shown as the mean ± SD of five independent donors (at least 120 follicles for each experiment. p-values were determined by ANOVA followed by Bonferroni test. **p < 0.01 and ***p < 0.001 compared with control group. ( c ) Human hair follicles were pre-treated with 10 μg/ml RAGE-FC for 1 h followed by 200 ng/ml HMGB1 for 3 days. Representative confocal images of hair follicles stained with COX-1 (red) and mPGES-1 (green) are shown. 4′-6-diamidino-2-phenylindole (DAPI; blue) was used to counterstain the nuclei. On day 3, each hair follicle was frozen and cryosectioned (6 μm). Data are representative of three independent experiments. Scale bar = 50 μm.

Article Snippet: Depending on the experiment, the media were supplemented with different concentrations of recombinant human HMGB1 (HMGB1; R&D systems, Minneapolis, MN, USA) or HMGB1 with antibodies blocking receptor for advanced glycation end-products (RAGE-FC, R&D systems), anti-TLR2 (BioLegend, San Diego, CA, USA), or anti-TLR4 (eBiosicence, San Diago, CA, USA) neutralizing antibodies.

Techniques: Control, Staining

Journal: International Journal of Nanomedicine

Article Title: Enhancement of Psoriasis Treatment by Phellodendri Chinensis Cortex Carbon Dots (PCC-CDs) Through Modulation of the HMGB1/TLR4/MAPK/NF-κB Pathway

doi: 10.2147/IJN.S578399

Figure Lengend Snippet: PCC-CDs spray regulate the HMGB1/TLR4/MAPK/NF-κB signaling pathway to ameliorate psoriasis-like dermatitis. ( A ) Serum levels of HMGB1, IFN-γ, and VEGF in mice. ( B ) Protein expression levels of HMGB1/TLR4/MAPK/NF-κB signaling pathway-related proteins in mouse skin tissues. Each group n = 3. Data were expressed as means ± standard deviation (SD). # P < 0.05, ## P < 0.01, ### P < 0.001 vs Control group; * P < 0.05, ** P < 0.01, *** P < 0.001vs Model group.

Article Snippet: The following antibodies were used: PCNA antibody (Cat. 10205-2-AP, Proteintech, Wuhan, China), Ki67 Polyclonal antibody (Cat. 28074-1-AP, Proteintech, Wuhan, China), HMGB1 antibody (Cat. 10829-1-AP, Proteintech, Wuhan, China), TLR4 antibody (Cat. 30400-1-AP, Proteintech, Wuhan, China), phospho-p38 MAPK (Thr180/Tyr182) Polyclonal antibody (Cat. 28796-1-AP, Proteintech, Wuhan, China), p38 MAPK Polyclonal antibody (Cat. 14064-1-AP, Proteintech, Wuhan, China), phospho-NF-κB p65 (Ser468) Recombinant antibody (Cat. 82335-1-AP, Proteintech, Wuhan, China), NF-κB p65 Polyclonal antibody (Cat. 10745-1-AP, Proteintech, Wuhan, China), GAPDH antibody (Cat. 60004-1-Ig, Proteintech, Wuhan, China), HRP-conjugated Goat Anti-Rabbit lgG (Cat. SA00001-2, Proteintech, Wuhan, China), HRP-conjugated Goat Anti-Mouse lgG (Cat. SA00001-1, Proteintech, Wuhan, China), Pentobarbital sodium salt (Cat. P3761, Sigma-Aldrich, St. Louis, MO,USA).

Techniques: Expressing, Standard Deviation, Control

Journal: International Journal of Nanomedicine

Article Title: Enhancement of Psoriasis Treatment by Phellodendri Chinensis Cortex Carbon Dots (PCC-CDs) Through Modulation of the HMGB1/TLR4/MAPK/NF-κB Pathway

doi: 10.2147/IJN.S578399

Figure Lengend Snippet: PCC-CDs alleviate M1 macrophage polarisation by modulating the HMGB1/TLR4/NF-κB signalling pathway. ( A ) Cytokine levels in cell supernatants, including HMGB1, TNF-α, IL-1β, IL-6 and IL-10. ( B ) The effects of PCC-CD intervention on HMGB1/TLR4/NF-κB protein expression in M1 macrophages (n = 3 per group). Data are expressed as mean ± standard deviation (SD). # P < 0.05, ## P < 0.01 compared with the control group; *P < 0.05, **P < 0.01 compared with the model group.

Article Snippet: The following antibodies were used: PCNA antibody (Cat. 10205-2-AP, Proteintech, Wuhan, China), Ki67 Polyclonal antibody (Cat. 28074-1-AP, Proteintech, Wuhan, China), HMGB1 antibody (Cat. 10829-1-AP, Proteintech, Wuhan, China), TLR4 antibody (Cat. 30400-1-AP, Proteintech, Wuhan, China), phospho-p38 MAPK (Thr180/Tyr182) Polyclonal antibody (Cat. 28796-1-AP, Proteintech, Wuhan, China), p38 MAPK Polyclonal antibody (Cat. 14064-1-AP, Proteintech, Wuhan, China), phospho-NF-κB p65 (Ser468) Recombinant antibody (Cat. 82335-1-AP, Proteintech, Wuhan, China), NF-κB p65 Polyclonal antibody (Cat. 10745-1-AP, Proteintech, Wuhan, China), GAPDH antibody (Cat. 60004-1-Ig, Proteintech, Wuhan, China), HRP-conjugated Goat Anti-Rabbit lgG (Cat. SA00001-2, Proteintech, Wuhan, China), HRP-conjugated Goat Anti-Mouse lgG (Cat. SA00001-1, Proteintech, Wuhan, China), Pentobarbital sodium salt (Cat. P3761, Sigma-Aldrich, St. Louis, MO,USA).

Techniques: Expressing, Standard Deviation, Control

Journal: International Journal of Nanomedicine

Article Title: Enhancement of Psoriasis Treatment by Phellodendri Chinensis Cortex Carbon Dots (PCC-CDs) Through Modulation of the HMGB1/TLR4/MAPK/NF-κB Pathway

doi: 10.2147/IJN.S578399

Figure Lengend Snippet: Schematic illustration of the therapeutic mechanism hypothesis of PCC-CDs spray in alleviating psoriatic inflammation via modulation of the HMGB1/TLR4/MAPK/NF-κB signaling pathway.

Article Snippet: The following antibodies were used: PCNA antibody (Cat. 10205-2-AP, Proteintech, Wuhan, China), Ki67 Polyclonal antibody (Cat. 28074-1-AP, Proteintech, Wuhan, China), HMGB1 antibody (Cat. 10829-1-AP, Proteintech, Wuhan, China), TLR4 antibody (Cat. 30400-1-AP, Proteintech, Wuhan, China), phospho-p38 MAPK (Thr180/Tyr182) Polyclonal antibody (Cat. 28796-1-AP, Proteintech, Wuhan, China), p38 MAPK Polyclonal antibody (Cat. 14064-1-AP, Proteintech, Wuhan, China), phospho-NF-κB p65 (Ser468) Recombinant antibody (Cat. 82335-1-AP, Proteintech, Wuhan, China), NF-κB p65 Polyclonal antibody (Cat. 10745-1-AP, Proteintech, Wuhan, China), GAPDH antibody (Cat. 60004-1-Ig, Proteintech, Wuhan, China), HRP-conjugated Goat Anti-Rabbit lgG (Cat. SA00001-2, Proteintech, Wuhan, China), HRP-conjugated Goat Anti-Mouse lgG (Cat. SA00001-1, Proteintech, Wuhan, China), Pentobarbital sodium salt (Cat. P3761, Sigma-Aldrich, St. Louis, MO,USA).

Techniques:

Journal: Molecular medicine reports

Article Title: Chrysin protects human osteoarthritis chondrocytes by inhibiting inflammatory mediator expression via HMGB1 suppression.

doi: 10.3892/mmr.2018.9724

Figure Lengend Snippet: Figure 4. Validation of HMGB‑1 levels in OA human chondrocyte model cells. Following CH treatment, HMGB‑1 was silenced by HMGB‑1‑specific siRNA or transfected with NC siRNA. (A) HMGB1 expression in whole cells from each group. (B) HMGB1 expression in the nucleus of each group. (C) HMGB1 immunofluo rescence was observed in cells under a laser scanning confocal microscope. Regions with HMGB‑1 are green; cell nuclei were stained with DAPI. Magnification, x400, scale bar=20 µm. CH, chrysin; HMGB‑1, high‑mobility group box chromosomal protein-1; siRNA, small interfering RNA; NC, negative control.

Article Snippet: The cells were incubated with anti‐human HMGB‐1 (1:2,000; cat no. M‐1702‐100; Biosensis Pty Ltd., Thebarton, Australia) at 4 ̊C overnight, followed by incubation with goat anti-human immunoglobulin G conjugated to Cy3 (1:400; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 37 ̊C for 1 h. Nuclei were counter stained with DAPI (1:1,000; Sigma‐Aldrich; Merck KGaA) for 5 min at room temperature.

Techniques: Biomarker Discovery, Transfection, Expressing, Microscopy, Staining, Small Interfering RNA, Negative Control

Journal: The FASEB Journal

Article Title: High‐mobility group box‐1 and inter‐alpha inhibitor proteins: In vitro binding and co‐localization in cerebral cortex after hypoxic‐ischemic injury

doi: 10.1096/fj.202002109rr

Figure Lengend Snippet: FIGURE 1 Direct physical binding between IAIPs and HMGB1: Solid-phase binding assays. A, The binding affinity of HMGB1 to IAIPs in vitro. The IAIPs were immobilized in the plate, solid bars, 200 ng per well, casein, open bars, 200 ng per well, as a negative control, and anti-HMGB1 antibody, hatched bars, 100 ng per well as a positive control, and increasing concentrations of the biotin-conjugated HMGB1 were sequentially added to the wells. The optical density plotted on the y-axis showed a concentration dependent increases in the binding of HMGB1 on the x-axis to the immobilized IAIPs. Values are mean ± standard deviation. B, The binding affinity of IAIPs to HMGB1 in vitro. The HMGB1 was immobilized in the plate, solid bars, 200 ng per well, casein, open bars, 200 ng per well as a negative control, and anti-IAIPs antibody, hatched bars, 0.7 ng per well as a positive control, and increasing concentrations of biotin-conjugated IAIPs were sequentially added to the wells. The optical density plotted on the y-axis showed a concentration dependent increases in the binding of IAIPs on the x-axis to the immobilized HMGB1. Values are mean ± standard deviation. C, The binding affinity of HMGB1 to bikunin, the light chain of IAIPs in vitro. Bikunin was immobilized in the plate, solid bars, 200 ng per well, casein, open bars, 200 ng per well, as a negative control, and anti-HMGB1 antibody, hatched bars, 100 ng per well, as a positive control, and sequential concentrations of the biotin-conjugated HMGB1 on the x-axis. The optical density of HMGB1 bound to bikunin, solid bars, was lower than the negative control, open bars, suggesting that HMGB1 did not bind to bikunin. Values are mean ± standard deviation

Article Snippet: Mouse anti- HMGB1 monoclonal antibody (Mab- 1690) was obtained from R&D Systems.

Techniques: Binding Assay, In Vitro, Negative Control, Positive Control, Concentration Assay, Standard Deviation

Journal: The FASEB Journal

Article Title: High‐mobility group box‐1 and inter‐alpha inhibitor proteins: In vitro binding and co‐localization in cerebral cortex after hypoxic‐ischemic injury

doi: 10.1096/fj.202002109rr

Figure Lengend Snippet: FIGURE 2 Direct physical binding between IAIPs and HMGB1: Surface plasmon resonance (SPR). A, The binding affinity of HMGB1 to IAIPs in vitro. Purified IAIPs (50 μg/mL) from human plasma immobilized on a CM5 Biacore chip, and sequential concentrations of the HMGB1 were passed over the chip. SPR showed a rapid increase in response units (RU), indicating the binding of HMGB1 to the immobilized IAIPs. The dissociation constants (Kd) for HMGB1 binding to IAIPs was 50.35 nmol/L. B, The binding affinity of IAIPs to HMGB1 in vitro. HMGB1 (5 μg/mL) was immobilized on a CM Biacore chip, and sequential concentrations of the IAIPs were passed over the chip. SPR showed a rapid increase in RU, indicating the binding of IAIPs to the immobilized HMGB1. The Kd for IAIPs binding to HMGB1 was 206.6 nmol/L

Article Snippet: Mouse anti- HMGB1 monoclonal antibody (Mab- 1690) was obtained from R&D Systems.

Techniques: Binding Assay, SPR Assay, In Vitro, Purification, Clinical Proteomics

Journal: The FASEB Journal

Article Title: High‐mobility group box‐1 and inter‐alpha inhibitor proteins: In vitro binding and co‐localization in cerebral cortex after hypoxic‐ischemic injury

doi: 10.1096/fj.202002109rr

Figure Lengend Snippet: FIGURE 3 Immunohistochemical expression of IAIPs and HMGB1 in cerebral cortices of neonatal rats in the Sham, and 0 and 3 h after exposure to HI in different magnification (40×, 100×). Representative images of IAIPs (Green fluorescence: A, E, I, M, Q, U), HMGB1 (Red fluorescence: B, F, J, N, R, V), DAPI (blue fluorescence: C, G, K, O, S, W), and merged double immunostaining (D, H, L, P, T, X) in cerebral cortex of neonatal rats in Sham 40× (A-D) and 100× (E-H), 0 h after exposure to HI 40× (I-L) and 100× (M-P), and 3 h after exposure to HI 40× (Q-T) and 100× (U-X). Scale bar = 50μm (40×) and 25 μm (100×). Each inset in 40× images shows the high magnification images below. White arrows in high magnification images designate the same cells in Sham (E-H), 0 (M-P), and 3 h (U-X) after HI, respectively. IAIPs appear to be predominantly localized to cytoplasm in Sham and 3 h after HI (Arrows, E, U), whereas mainly localized to nucleus at 0 h after HI (Arrow, M). HMGB1 appears to be predominantly localized to the nucleus in Sham and 0 h after HI (Arrows, F and N), whereas mainly localized to cytoplasm at 3 h after HI (Arrow, V)

Article Snippet: Mouse anti- HMGB1 monoclonal antibody (Mab- 1690) was obtained from R&D Systems.

Techniques: Immunohistochemical staining, Expressing, Fluorescence, Double Immunostaining

Journal: The FASEB Journal

Article Title: High‐mobility group box‐1 and inter‐alpha inhibitor proteins: In vitro binding and co‐localization in cerebral cortex after hypoxic‐ischemic injury

doi: 10.1096/fj.202002109rr

Figure Lengend Snippet: FIGURE 5 Quantification of IAIP and HMGB1 immunofluorescent staining and co-localization of IAIP-HMGB1 in the cells of the cerebral cortices in the neonatal rats. Graphs representing the IAIPs (A, B, C), HMGB1 (D, E, F), and IAIPs-HMGB1 merged (G, H, I) expressed as the percent of DAPI positive cells plotted on the y-axis for total (A, D, G), cytoplasmic (B, E, H), and nuclear (C, F, I) compartments in cerebral cortices of neonatal rats for the Sham, HI-0 h, and HI-3 h groups on the x-axis. Ten fields per slide per animal, Sham (n = 6), HI-0 h (n = 6) and HI-3 (n = 5) groups were analyzed. Statistical analysis by one-way analysis of variance (ANOVA), Turkey's post hoc test, *P < .05, **P < .01, ***P < .001. Values are median and scatter grams

Article Snippet: Mouse anti- HMGB1 monoclonal antibody (Mab- 1690) was obtained from R&D Systems.

Techniques: Staining

Journal: The FASEB Journal

Article Title: High‐mobility group box‐1 and inter‐alpha inhibitor proteins: In vitro binding and co‐localization in cerebral cortex after hypoxic‐ischemic injury

doi: 10.1096/fj.202002109rr

Figure Lengend Snippet: FIGURE 6 Correlational analyses between the percent of IAIP and HMGB1 positive cells. A, Ratio of IAIPs on the y-axis plotted for the ratio of HMGB1 as a percent of DAPI positive cells on the x-axis for the total, cytoplasmic, and nuclear compartments. Open circles are Sham control (n = 6), solid circles HI-0 h (n = 6), and solid triangles HI-3 h (n = 5) groups. Solid line designates the regression line. Dashed lines are the 95% confidence intervals. Statistical analysis by Pearson product-moment correlation coefficients. B, Ratio of IAIPs on the y-axis plotted for the ratio of HMGB1 as a percent of DAPI positive cells on the x-axis for the total, cytoplasmic, and nuclear compartments. Solid circles the HI-0 h (n = 6) and solid triangles the HI-3 h (n = 5) groups. Solid line designates the regression line. Statistical analysis by Pearson product-moment correlation coefficients. Dashed lines are the 95% confidence intervals. Statistical analysis by Pearson product-moment correlation coefficients

Article Snippet: Mouse anti- HMGB1 monoclonal antibody (Mab- 1690) was obtained from R&D Systems.

Techniques: Control

Journal: Clinical Proteomics

Article Title: Dimethyl fumarate alleviates inflammation during high altitude hypoxia induced acute lung injury by upregulating Nrf2/SLC7A11 pathway in ferroptosis

doi: 10.1186/s12014-025-09566-0

Figure Lengend Snippet: Effects of DMF against ferroptosis of lung epithelial cells was abolished in SLC7A11 knock-down BEAS-2B cells and reversed in SLC7A11 overexpression BEAS-2B cells. (A) Impacts of varying DMF concentrations on the viability of BEAS-2B cells. (B) Nrf2 in the nucleus was significantly reduced under hypoxia exposure, which was then increased by the DMF pretreatment. ROS flow cytometry (C) , ROS immunofluorescence (D) , GSH levels, MDA, Fe 2+ and SOD activity (E) , and ferroptosis-related protein and mRNA expression (F , G) in BEAS-2B cells pre-treated with DMF or SLC7A11 knock-down. SLC7A11 and GPX4 protein expression (H) , ROS immunofluorescence (J) , Fe 2+ , SOD activity and GSH levels (K-M) in BEAS-2B cells pre-treated with DMF and SLC7A11 overexpression. (I) Hypoxia exposure significantly increased the expression and secretion of HMGB1, while DMF pretreatment could alleviate its expression. ( n = 3). Hypoxia-co-culture group: hypoxic co-culture for 48 h, hypoxic ALI group; Hypoxia-co-DMF group: DMF pre-treated for 24 h and hypoxic co-culture for 48 h. * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Article Snippet: The membrane was then blocked for 1 h in a solution of 5% heated, filtered skimmed milk in TBS-T, then incubated overnight with the primary antibodies of glutathione peroxidase 4 (GPX4), Solute Carrier Family 7 Member 11 (SLC7A11), ferritin heavy chain 1 (FTH1), acyl-CoA synthetase long chain family member 4 (ACSL4), interleukin-6 (IL-6), IL-1, tumor necrosis factor-α (TNF-α), HMGB1, and β-actin separately (CST, America) (dilution 1:1000) at 4 °C.

Techniques: Knockdown, Over Expression, Flow Cytometry, Immunofluorescence, Activity Assay, Expressing, Co-Culture 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, Gene Expression, 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, Protein-Protein interactions, 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: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

doi: 10.1161/ATVBAHA.120.314557

Figure Lengend Snippet: Figure 1. xxxx. A, HMGB1 (high mobility group box 1) was knocked down by siRNA in human coronary artery endothelial cells (HCAECs) achieving a reduction of 80% to 90% in protein levels. Representative blots (left); quantification (right). n=4 independent experiments; ***P<0.001 by t test. Error bars represent SEM. B, Transcytosis of DiI-tagged LDL (low-density lipoprotein) across confluent HCAEC from male donors is measured in real time using total internal reflection fluorescence microscopy. Knockdown of HMGB1 led to a decrease in LDL transcytosis; each point represents 1 cell. n=4 experiments; ***P<0.001 by t test; error bars represent SD. C, Knockdown of HMGB1 led to a decrease in LDL transcytosis across confluent cells from female donors; each point represents 1 cell. n=4 experiments; ***P<0.001 by t test; error bars represent SD. D, Knockdown of HMGB1 had no effect on albumin transcytosis. Transcytosis of Alexa 488–albumin across confluent cells from a male donor was measured; each point represents 1 cell. n=4 experiments; error bars represent SD. E, Knockdown of HMGB1 in human coronary artery endothelial cells had no effect on transendothelial electrical resistance (ER); PAF (platelet-activating factor) is a positive control. n=4; ***P<0.001 by 1-way ANOVA; ns indicates nonsignificant by Tukey multiple comparison test.

Article Snippet: After washing, cells were stained with 1:25 (36 μg/mL) Alexa Fluor 405 anti-HMGB1 antibody (NB100-2322AF405; Novus) in 2% FBS in PBS for 15 minutes on ice.

Techniques: Fluorescence, Microscopy, Knockdown, Positive Control, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

doi: 10.1161/ATVBAHA.120.314557

Figure Lengend Snippet: Figure 3. xxxx. A, HMGB1 (high mobility group box 1) knockdown in human coronary artery endothelial cells by siRNA led to a decrease in both mature (m) and precursor (p) forms of SREBP2 (sterol regulatory element-binding protein 2) by Western blot. Representative blot (left); quantification (right). n=4; *P<0.05 by t test; error bars represent SEM. B, Knockdown of either HMGB1 or SREBP2 significantly inhibited LDL (low-density lipoprotein) transcytosis, but depletion of both did not achieve any further reduction; each point represents 1 cell. n=4 experiments; ***P<0.001 by 1-way ANOVA; error bars represent SD. C, SREBP2 was knocked down by siRNA achieving a reduction in protein levels of around 80% (quantification is of precursor form). n=4; *P<0.05 by t test; error bars represent SEM. D, Depletion of SREBP2 prevented induction of LDL transcytosis caused by HMGB1 overexpression. Each point represents 1 cell. n=4 experiments; ***P<0.001 by 1-way ANOVA; error bars represent SD; ns indicates nonsignificant by Tukey multiple comparison test.

Article Snippet: After washing, cells were stained with 1:25 (36 μg/mL) Alexa Fluor 405 anti-HMGB1 antibody (NB100-2322AF405; Novus) in 2% FBS in PBS for 15 minutes on ice.

Techniques: Knockdown, Binding Assay, Western Blot, Over Expression, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

doi: 10.1161/ATVBAHA.120.314557

Figure Lengend Snippet: Figure 4. xxxx. A, Human coronary artery endothelial cells (HCAECs) were immunostained for HMGB1 (high mobility group box 1; red) before and after incubation with LDL (low-density lipoprotein; 40 μg/mL) for 15 min. LDL significantly increased the nuclear-to-cytoplasmic ratio, and SR-BI (scavenger receptor class B type 1) depletion by siRNA partially inhibited this effect. Representative images (left); quantification (right). n=4; ***P<0.001 by 1-way ANOVA, ***P<0.001 and *P<0.05 by Tukey multiple comparison test; error bars represent SEM. Scale bars=30 µm. B, FRAP of nuclear HMGB1 in HCAECs expressing GFP-HMGB1 in the presence or absence of 40 µg/mL unlabeled LDL; ***P<0.001 by Holm-Sidak multiple comparisons test for each time point. Error bars represent SD. C, Incubation of serum-starved endothelial cells with LDL (40 μg/mL, 24 h) induced HMGB1 expression. Western blot is representative of 4 independent experiments; **P<0.01 by paired t test with data normalized to control. D, The stability of SREBP2 (sterol regulatory element-binding protein 2) mRNA was assessed in HCAECs. The half-life of SREBP2 mRNA in control cells and cells depleted of HMGB1 by siRNA was measured in the presence of actinomycin. E, The half-life of SREBP2 protein was assessed in cells depleted of HMGB1 by siRNA; cycloheximide (CHX) was used to inhibit protein synthesis for the indicated number of hours (h). *P<0.05 and **P<0.01 by Holm-Sidak multiple comparisons test for each time point. n=4 experiments; error bars represent SEM. M indicates mature; and p, precursor.

Article Snippet: After washing, cells were stained with 1:25 (36 μg/mL) Alexa Fluor 405 anti-HMGB1 antibody (NB100-2322AF405; Novus) in 2% FBS in PBS for 15 minutes on ice.

Techniques: Incubation, Comparison, Expressing, Western Blot, Control, Binding Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

doi: 10.1161/ATVBAHA.120.314557

Figure Lengend Snippet: Figure 5. xxxx. A, Mice deficient in endothelial HMGB1 (high mobility group box 1) and wild-type littermates were injected retro-orbitally with 200 μg of AlexaFluor (AF) 568–conjugated LDL (low-density lipoprotein) and euthanized 30 min later. The accumulation of LDL was measured and normalized to the imaged area and to the paired control animal. Representative images of the inner curvature of the aorta en face (left); quantification with each point representing 1 animal (right); *P<0.05 by paired, nonparametric t test. B, Mice deficient in endothelial HMGB1 and wild-type littermates were fed a high-fat diet for 18 wk followed by staining of the inner curvature of the aorta with oil red O (ORO). Of 12 wild-type mice, 6 developed visible plaque compared with 5 (of 11) knockout mice. Of those displaying plaque, endothelial cell (EC)–HMGB1−/− mice showed significantly smaller fatty streaks than littermate controls (scatterplot). Representative images (left); quantification (right); each point represents 1 animal; **P<0.01 by t test. C, Lipid accumulation at the aortic root was also measured (representative image at left) and quantified (right); data are from 7 knockout animals and 9 controls. D, EC-HMGB1−/− mice and controls were injected via retro-orbital vein with 1% Evans blue dye and then euthanized after 30 min. Permeability to Evans blue dye was not significantly altered. n=4 to 5. ns indicates nonsignificant.

Article Snippet: After washing, cells were stained with 1:25 (36 μg/mL) Alexa Fluor 405 anti-HMGB1 antibody (NB100-2322AF405; Novus) in 2% FBS in PBS for 15 minutes on ice.

Techniques: Injection, Control, Staining, Knock-Out, Permeability

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

doi: 10.1161/ATVBAHA.120.314557

Figure Lengend Snippet: Figure 6. xxxx. A, Endothelial cell (EC)–HMGB1−/−:LDLR−/− and LDLR−/− littermate controls were injected retro-orbitally with 200 μg of AlexaFluor (AF) 568–conjugated LDL (low-density lipoprotein) and euthanized 30 min later. The accumulation of LDL was measured and normalized to the imaged area and to the paired control animal. Representative images of the inner curvature of the aorta en face (left); quantification with each point representing 1 animal (right); *P<0.05 by paired, nonparametric t test. B, Double knockout mice (EC-HMGB1−/−:LDLR−/−) and LDLR−/− littermate controls were fed a high-fat diet for 5 wk followed by staining of the inner curvature of the aorta with oil red O (ORO). EC-HMGB1−

Article Snippet: After washing, cells were stained with 1:25 (36 μg/mL) Alexa Fluor 405 anti-HMGB1 antibody (NB100-2322AF405; Novus) in 2% FBS in PBS for 15 minutes on ice.

Techniques: Injection, Control, Double Knockout, Staining

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: Fig. 1. 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: Fig. 2. 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: Fig. 3. 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: Fig. 5. 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: Fig. 6. 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: Fig. 7. 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: Fig. 8. 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: Fig. 9. 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: Leukemia

Article Title: HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

doi: 10.1038/leu.2010.225

Figure Lengend Snippet: Figure 1 Inhibition of HMGB1 release increases chemotherapy sensitivity in leukemia cells. (a) HL-60 and Jurkat cells were treated with VCR (1 mg/ml), ADM (1 mg/ml), cytosine arabinoside (Ara-C, 0.2 mM) or arsenic trioxide (AS2O3, 5 mM) for 24 h and then the cell viability (top) and HMGB1 release (bottom) was analyzed (n ¼ 3, *Po0.05 versus control group). Cell viability of control was set as 100%. (b, c) HL-60 and Jurkat cells were treated with VCR (1 mg/ml) or ADM (1 mg/ml) for 0–48 h with or without HMGB1-neutralizing antibody (‘b’, 10 mg/ml) or quercetin (‘c’, 50 mM), and then cell viability was analyzed (n ¼ 3, *Po0.05 versus control group). Cell viability of control was set as 100%. (d) After transfection with HMGB1 shRNA or control shRNA for 48 h, cells, as indicated, were treated with VCR (1 mg/ml), ADM (1 mg/ml), cytosine arabinoside (Ara-C, 0.2 mM) or arsenic trioxide (AS2O3, 5 mM) for 24 h, then cell viability was analyzed (n ¼ 3, *Po0.05 versus control shRNA group). Cell viability of control was set as 100%.

Article Snippet: The antibodies to HMGB1, LC3 and p62 were obtained from Novus (Littleton, CO, USA).

Techniques: Inhibition, Control, Transfection, shRNA

Journal: Leukemia

Article Title: HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

doi: 10.1038/leu.2010.225

Figure Lengend Snippet: Figure 2 Autophagy is required for exogenous HMGB1-mediated chemotherapy resistance. (a) HL-60 and Jurkat cells were pre-treated with HMGB1 (1 mg/ml) for 24 h and then treated with VCR (1 mg/ml), ADM (1 mg/ml), cytosine arabinoside (Ara-C, 0.2 mM) or arsenic trioxide (AS2O3, 5 mM) for 48 h, then cell viability was analyzed (n ¼ 3, *Po0.05). Cell viability of control was set as 100%. (b) After transfection with Beclin1 shRNA or control shRNA for 48 h, cells were treated with HMGB1 protein (1 mg/ml) for 24 h and then treated with VCR (1 mg/ml) or ADM (1 mg/ml) for 48 h, then cell viability was analyzed (n ¼ 3, *Po0.05). Cell viability of control was set as 100%. (c) HL-60 cells were treated with HMGB1 protein (1 mg/ml, 24 h) with or without bafilomycin A1 (‘Baf A1’, 100 nM) and then treated with VCR (1 mg/ml), ADM (1 mg/ml), cytosine arabinoside (Ara-C, 0.2 mM) or arsenic trioxide (AS2O3, 5 mM) for 48 h, then cell viability was analyzed (n ¼ 3, *Po0.05). Cell viability of control was set as 100%.

Article Snippet: The antibodies to HMGB1, LC3 and p62 were obtained from Novus (Littleton, CO, USA).

Techniques: Control, Transfection, shRNA

Journal: Leukemia

Article Title: HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

doi: 10.1038/leu.2010.225

Figure Lengend Snippet: Figure 3 HMGB1 induces autophagy in leukemia cells. (a) HL-60 and Jurkat cells were treated with HMGB1 (1 mg/ml) for 6–48 h. The total protein extracts were used for western blot analysis (n ¼ 3, *Po0.05 versus control group). ‘AU’: arbitrary units. (b) Analysis of LC3 and p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (‘PA’, 10 mg/ml) and E64D (10 mg/ml) after HMGB1 (1 mg/ml) treatment for 48 h (n ¼ 3, *Po0.05). ‘AU’: arbitrary units. (c) HL-60 cells were treated with HMGB1 (1 mg/ml) for 48 h. The LC3 punctae formation was assayed by confocal microscopic analysis (*Po0.05). Bar ¼ 10 mm. (d) HMGB1 was knocked down in HL-60 and Jurkat using shRNA for 48 h, and then these cells were treated with VCR (1 mg/ml), ADM, (1 mg/ml) or arsenic trioxide (AS2O3, 5 mM) for 24 h. The LC3 punctae formation was assayed by confocal microscopic analysis (*Po0.05 versus control shRNA group). (e, f) HL-60 cells were treated with HMGB1 (10 mg/ml) for 6 h, then cell lysates were prepared for immunoprecipitation (‘IP’) as indicated. The resulting immune complexes and inputs were analyzed by western blotting (‘IB’) as indicated. The cells were cultured in Hank’s balanced salt solution (starvation) for 2 h as a positive control. (g) HL-60 cells were treated with HMGB1 (1 mg/ml) for 48 h. The colocalization of LC3 (green) and Atg5 (red) was assayed by confocal microscopic analysis (*Po0.05). Bar ¼ 10 mm. (h) Ultrastructural features in HL-60 cells with or without HMGB1 (1 mg/ml, 48 h) treatment (‘AP’: autophagosomes, ‘AL’: autolysosomes) (*Po0.05). Bar ¼ 0.5 mm.

Article Snippet: The antibodies to HMGB1, LC3 and p62 were obtained from Novus (Littleton, CO, USA).

Techniques: Western Blot, Control, shRNA, Immunoprecipitation, Cell Culture, Positive Control

Journal: Leukemia

Article Title: HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

doi: 10.1038/leu.2010.225

Figure Lengend Snippet: Figure 4 PI3K-III is required for HMGB1-induced autophagy. (a) Analysis of LC3 and p62 by western blot in the presence or absence of ‘3-MA’ (10 mM) after HMGB1 (1 mg/ml) treatment for 48 h (n ¼ 3, *Po0.05). ‘AU’: arbitrary units. (b) After transfection with PI3K- III shRNA or control shRNA for 48 h, cells were treated with HMGB1 protein (1 mg/ml) for 48 h. The total protein extracts were used for western blot analysis (n ¼ 3, *Po0.05). ‘AU’: arbitrary units.

Article Snippet: The antibodies to HMGB1, LC3 and p62 were obtained from Novus (Littleton, CO, USA).

Techniques: Western Blot, Transfection, shRNA, Control

Journal: Leukemia

Article Title: HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

doi: 10.1038/leu.2010.225

Figure Lengend Snippet: Figure 5 MEK–ERK pathway is required for HMGB1-induced autophagy. (a) Analysis of LC3 and p62 by western blot in the presence or absence of ‘3-MA’ (10 mM) or UO126 (20 mM) after HMGB1 (1 mg/ml) treatment for 48 h (n ¼ 3, *Po0.05). ‘AU’: arbitrary units. (b) After transfection with MEK1/2 siRNA or control siRNA for 48 h, cells were treated with HMGB1 protein (1 mg/ml) for 48 h. The total protein extracts were used for western blot analysis (n ¼ 3, *Po0.05). ‘AU’: arbitrary units. (c, d) After transfection with shRNA as indicated for 48 h, HL-60 cells were treated with VCR (1 mg/ml, 24 h), ADM (1 mg/ml, 24 h) or HMGB1 (1 mg/ml, 48 h), then the LC3 punctae formation was assayed by confocal microscopic analysis (*Po0.05). (e) Ultrastructural features in indicated HL-60 cells with or without HMGB1 (1 mg/ml, 48 h). The number of autophagosomes under transmission electron microscope was calculated (*Po0.05 versus control shRNA group). Representative image with HMGB1 treatment is shown in left panel. Bar ¼ 2 mm.

Article Snippet: The antibodies to HMGB1, LC3 and p62 were obtained from Novus (Littleton, CO, USA).

Techniques: Western Blot, Transfection, Control, shRNA, Transmission Assay, Microscopy

Journal: Leukemia

Article Title: HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells.

doi: 10.1038/leu.2010.225

Figure Lengend Snippet: Figure 6 Schematic of the mechanism by which HMGB1 modulates drug resistance by regulating autophagy. HMGB1 is released in leukemia cell lines after chemotherapy-induced cytotoxicity. The released HMGB1 functions as an activator of autophagy, which increases drug resistance through the PI3K–MEK–ERK pathway. Knockdown by RNA interference or pharmacological inhibition (for example, 3-MA and U0126) of the PI3K–MEK–ERK pathway reverses the resistance of leukemia cells to chemotherapy.

Article Snippet: The antibodies to HMGB1, LC3 and p62 were obtained from Novus (Littleton, CO, USA).

Techniques: Knockdown, Inhibition