Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway

doi: 10.3389/fphar.2020.598939

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway

doi: 10.3389/fphar.2020.598939

Figure Lengend Snippet: TLR4 and NF-κB were over expressed in breast cancer and correlated with TNM stages. (A, B) Representative immunohistochemistry staining for TLR4 and NF-κB p65 in normal breast tissues and breast cancer tissues with different TNM stages. * p < 0.05, ** p < 0.01 between different groups. (C, D) The expression of TLR4, p-NF-κB p65 and NF-κB p65 in MCF 10A, MCF-7 and MDA-MB-231 cells by western blot. Significant differences between different groups were indicated as * p < 0.01, vs. MCF 10A group, n = 3.

Article Snippet: The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used.

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway

doi: 10.3389/fphar.2020.598939

Figure Lengend Snippet: AT-I regulated TLR4/NF-κB signaling pathway in breast cancer cells. (A, B) The effects of AT-I on TLR4/NF-κB signaling pathway were measured by western blot assay. (C, D) The levels of TNF-α, IL-6 and IL-1β in the supernatants of cells were measured by ELISA assay. Significant differences between different groups were indicated as * p < 0.05, ** p < 0.01, vs. the 0 μM control group or the LPS treated control group, n = 3.

Article Snippet: The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used.

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

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway

doi: 10.3389/fphar.2020.598939

Figure Lengend Snippet: The effects of AT-I on suppressing tumorigenesis was mediated by TLR4. The shTLR4 plasmid was used to transfect breast cancer cells, and then western blot and ELISA assays were used to compare related proteins expression with untransfected cells after cells had been pre-treated in presence or absence AT-I for 48 h during LPS stimulation. (A, B) The expression of TLR4/NF-κB signaling pathway was detected by western blot assay. (C, D) The levels of TNF-α, IL-6 and IL-1β in the cell supernatants were measured by ELISA assay. Significant differences between different groups were indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the LPS treated control group, n = 3.

Article Snippet: The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used.

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

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway

doi: 10.3389/fphar.2020.598939

Figure Lengend Snippet: The effects of AT-I on cell migration and invasion was mediated by TLR4. LPS induced shTLR4 transfected and control cells were treated with AT-I, and then seeded in 24-well plates or transwell chamber coated with matrigel. (A, B) The migration capability were calculated by wound healing assay (40×magnification). (C, D) The invasion capability were calculated by transwell invasion assay (100×magnification). Significant differences between different groups were indicated as * p < 0.05, ** p < 0.01, vs. the LPS treated control group, n = 3.

Article Snippet: The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used.

Techniques: Migration, Transfection, Control, Wound Healing Assay, Transwell Invasion Assay

Journal: Frontiers in Pharmacology

Article Title: Atractylenolide-I Suppresses Tumorigenesis of Breast Cancer by Inhibiting Toll-Like Receptor 4-Mediated Nuclear Factor-κB Signaling Pathway

doi: 10.3389/fphar.2020.598939

Figure Lengend Snippet: AT-I inhibited NMU-induced rat breast cancer. The NMU-induced rats were treated with control or AT-I for 9 weeks. (A) Body weights of rats in all groups at 0, 3, 5 and 9 weeks. (B) Palpable tumor incidence was recorded every week. (C) Average tumor number at 9 week. (D) Average tumor volume at 9 week. (E, F) Rat breast tissues were kept to detect TLR4/NF-κB pathway by western blot assay at the end of experiment. (G, H, I) TNF-α, IL-6 and IL-1β levels in rat breast tissues were analyzed by ELISA at the end of experiment. Significant differences between different groups were indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, vs. the NMU group, n = 6.

Article Snippet: The anti-TLR4 monoclonal antibody (1:100) and NF-κB p65 (1:100) were applied for 1 h at 37 °C, and SABC Staining System kit (Boster, Wuhan, China) containing secondary antibody was used.

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

Journal: The Journal of biological chemistry

Article Title: DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein.

doi: 10.1016/j.jbc.2022.102577

Figure Lengend Snippet: Figure 6. Potential roles of the DNA-mediated proteolytic processing of HMGB1 by neutrophil elastase in NETs. Due to the enhanced binding activities of the processed HMGB1 protein, this processing may promote (1) TLR4 signaling, (2) binding to biofilm DNA, and (3) DNA sensing by cGAS. Due to the loss of residues 177–215, the processing of HMGB1 may diminish (4) RAGE signaling and (5) nuclear localization. NET, neutrophil extracellular trap.

Article Snippet: Lyophilized TLR4 MD-2 complex was purchased from R&D Systems (catalog no.: #3146-TM-050).

Techniques: Binding Assay

Journal: Cell Death & Disease

Article Title: Dopamine D3 receptor signaling alleviates mouse rheumatoid arthritis by promoting Toll-like receptor 4 degradation in mast cells

doi: 10.1038/s41419-022-04695-y

Figure Lengend Snippet: A Changes in the signaling of p38, ERK, and JNK, as well as their downstream molecules AP-1 (c-jun) and NF-κB (p65), were tested by western blotting in a mouse CIA model. B ELISA was used to check the cytokines after mast cells were treated with signaling inhibitors to block their function. p38 inhibitor SB202190 20 μM, ERK inhibitor PD98059 10 μM, JNK inhibitor SP600125 20 μM, c-jun inhibitor T-5224 30 μM and p65 inhibitor JSH-23 10 μM. C Changes in TLR4 in the mouse knee joint were observed by western blotting. D Both D3R and TLR4 in p815 mast cells were silenced, and cytokines were measured. All data are representative of the means ± SEM. One-way ANOVA (Tukey’s post hoc), n = 3–4; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Antibodies against D3R (#sc-136170), GAPDH (#sc-47724), p-p38 (#4511), p-ERK (#4370), p-JNK (#9255), p-c-jun (#3270), p-p65 (#3033), TLR4 (#14358), ubiquitin (#3936), LC3-I/II (#12741), mTOR (#2983), p-mTOR (#2971), Akt (#4691), p-Akt (#4060), p-AMPK (#4186) and AMPK (#sc-398861) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Cell Death & Disease

Article Title: Dopamine D3 receptor signaling alleviates mouse rheumatoid arthritis by promoting Toll-like receptor 4 degradation in mast cells

doi: 10.1038/s41419-022-04695-y

Figure Lengend Snippet: A trl4 mRNA levels in joints were examined after CIA induction ( n = 3). B TLR4 expression was checked after mice were treated with the D3R agonist 7-OH ( n = 6). C TLR4 staining was exhibited by laser confocal microscopy images in p815 mast cells after D3R knockdown and LPS/DA exposure. The mean fluorescence intensity was measured by ImageJ software ( n = 3). D TLR4 expression was checked after ubiquitin (Ub) knockdown and 7-OH treatment upon LPS/DA exposure in p815 mast cells ( n = 3). E Ubiquitin content was examined after pulling down TLR4 in p815 mast cells ( n = 3). F The colocalization of TLR4 with ubiquitin was checked after 7-OH treatment in p815 mast cells ( n = 3). G and H LC3-II puncta ( n = 10) and LC3-II and p62 expression ( n = 3) were observed by laser confocal microscopy images or western blotting. Mean fluorescence intensity was used to represent the expression of LC3-II in p815 mast cells. I TLR4 expression was examined after LC3-II knockdown in p815 mast cells. J The colocalization of TLR4 with lysosomes was checked after LC3-II knockdown in p815 mast cells ( n = 5). K ELISA revealed proinflammatory cytokines in activated p815 mast cells after LC3-II knockdown and 7-OH treatment ( n = 4). All data are representative of the means ± SEM. Student’s t-test was used for ( F ), and one-way ANOVA (Tukey’s post hoc) was used for other panels; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar = 5 μm.

Article Snippet: Antibodies against D3R (#sc-136170), GAPDH (#sc-47724), p-p38 (#4511), p-ERK (#4370), p-JNK (#9255), p-c-jun (#3270), p-p65 (#3033), TLR4 (#14358), ubiquitin (#3936), LC3-I/II (#12741), mTOR (#2983), p-mTOR (#2971), Akt (#4691), p-Akt (#4060), p-AMPK (#4186) and AMPK (#sc-398861) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Cell Signaling Technology (Danvers, MA).

Techniques: Expressing, Staining, Confocal Microscopy, Knockdown, Fluorescence, Software, Ubiquitin Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Dopamine D3 receptor signaling alleviates mouse rheumatoid arthritis by promoting Toll-like receptor 4 degradation in mast cells

doi: 10.1038/s41419-022-04695-y

Figure Lengend Snippet: A – C After D3R knockdown, phosphorylated mTOR, AKT, and AMPK content was checked by laser confocal microscopy in p815 mast cells. Mean fluorescence intensity was used to represent the expression. D Phosphorylated mTOR, AKT, and AMPK were checked by western blotting after 7-OH treatment. E – G TLR4 expression was checked by western blotting after the p815 mast cells were treated with the AKT antagonist GDC0068 (1 μM), mTOR inhibitor rapamycin (50 μM), and AMPK agonist A769662 (100 μM) after D3R knockdown. H The cytokines in activated p815 mast cells were measured after above the antagonists and agonist treatment. I Schematic diagram showing how TLR4 degradation is regulated by D3R and potent signaling pathways. Student’s t-test was used for ( F ) and one-way ANOVA (Tukey’s post hoc) was used for other panels; *, p < 0.05; **, p < 0.01; ***, p < 0.001, n = 3. Scale bar = 5 μm.

Article Snippet: Antibodies against D3R (#sc-136170), GAPDH (#sc-47724), p-p38 (#4511), p-ERK (#4370), p-JNK (#9255), p-c-jun (#3270), p-p65 (#3033), TLR4 (#14358), ubiquitin (#3936), LC3-I/II (#12741), mTOR (#2983), p-mTOR (#2971), Akt (#4691), p-Akt (#4060), p-AMPK (#4186) and AMPK (#sc-398861) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Cell Signaling Technology (Danvers, MA).

Techniques: Knockdown, Confocal Microscopy, Fluorescence, Expressing, Western Blot, Protein-Protein interactions

Journal: Frontiers in Pharmacology

Article Title: Periplaneta americana extract alleviates steatohepatitis in a mouse model by modulating HMGB1-mediated inflammatory response

doi: 10.3389/fphar.2022.995523

Figure Lengend Snippet: GLC blocks M1 polarization by decreasing HMGB1 released by hepatocytes. (A) Immunofluorescence staining of HMGB I in mouse liver tissues. Scale bars, 50 μm. (B) Statistics of cytoplasmic HMGB1 positive cells of IF staining sections (C) Dataset (GEO database- GSE28619) of patients with alcoholic hepatitis were analyzed. Expression levels of M1 related markers (CD86, CXCL9, CXCL1 0 and TLR4) in HMGB1-High group and HMGB1-Low group. (D) Schematic flow chart of co-culture system. NCTC1469 cells induced by EtOH with or without treatment of GLC serum. Supemetant was collected for ELISA and culturing macrophages. The treatment of NCTC1469 cells was displayed in table (E) Supernatant HMGB1 levels of NCTC1469 cells induced by EtOH. ** P < 0.01 vs. the NCTC1469 cells with EtOH treatment. (F) Representative iNOS immunofluorescence staining and statistics of macrophages treated with condition medium (CM) of NCTC1469 cells. Scale bars, 50 μm. (G) Quantitative PCR analysis of J774A.1 cell cocultured with NCTCI469 cells. The data were obtained from three dependent experiments per group. * P < 0.05 vs. G#1, *** P < 0.001 vs. G#1.

Article Snippet: The membranes were washed, blocked and incubated with specific primary anti-rabbit antibodies against CYP2E1 (Proteintech, #19937-1-AP), GAPDH (Cell signaling Technology, #2118S), pNF-kB p65 (Wanleibio, #WL02169), NF-kB p65 (Wanleibio, #WL01273b), TLR4 (PTMBIO, #PTM-5192).

Techniques: Immunofluorescence, Staining, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Periplaneta americana extract alleviates steatohepatitis in a mouse model by modulating HMGB1-mediated inflammatory response

doi: 10.3389/fphar.2022.995523

Figure Lengend Snippet: GLC ameliorates progression of steatohepatitis via HMGB1-mediated TLR4/NF-KB pathway (A) Western blot analysis of indicated proteins expression. (B) The relative intensity of indicated proteins expression. The data were obtained from three dependent experiments per group. (C) Process of GLC inhibiting HMGB1-mediated macrophage activation by TLR4/NF-KB in the progression of steatohepatitis. Alcoholics and free fatty acids cause oxidative stress injury and lipid accumulation of hepatocytes. The dying/dead hepatocytes release DAMPs - HMGB1 to the extracellular. HMGB1 combined with TLR4 of macrophages to activate the inflammatory response through the NF-KB pathway, which aggravated the hepatocyte injury. GLC exerts hepatoprotective properties by reducing lipid accumulation and oxidative stress. It decreases the HMGB1 in extracellular and blocks TLR4/NF-KB mediated inflammatory cytokines expression of macrophages.

Article Snippet: The membranes were washed, blocked and incubated with specific primary anti-rabbit antibodies against CYP2E1 (Proteintech, #19937-1-AP), GAPDH (Cell signaling Technology, #2118S), pNF-kB p65 (Wanleibio, #WL02169), NF-kB p65 (Wanleibio, #WL01273b), TLR4 (PTMBIO, #PTM-5192).

Techniques: Western Blot, Expressing, Activation Assay