Journal: Immunobiology

Article Title: Giardia duodenalis triggered neutrophil extracellular traps in goats.

doi: 10.1016/j.imbio.2025.152894

Figure Lengend Snippet: Fig. 7. NETs release and ROS production was associated with TLR2/TLR4 (A) NETs quantitation. (B) ROS level. Goat neutrophils were pretreated with TLR2 in hibitor C29 (100 μM) and TLR4 inhibitor TLR4-IN-C34 (10 μM) respectively for 30 min, and stimulated with Giardia (ratio 1:1) for 2 h. Zymosan (1 mg/mL) was used as a positive control. Data were expressed as mean ± SD (n = 5). P < 0.05 was considered significant (***P < 0.001).

Article Snippet: In inhibition tests, prior to stimulation with G. duodenalis (at a ratio of 1:1), neutrophils were pretreated with 10 μM of the NADPH oxidase inhibitor Diphenyleneiodonium chloride (DPI, Sigma-Aldrich, USA), 50 μM of ERK1/2 inhibitor U0126 (Sigma-Aldrich, USA), 10 μM of the p38 MAPK signaling pathway inhibitor SB202190 (Sigma-Aldrich, USA), 100 μM of TLR2 inhibitor C29 (MedChemExpress, USA) and 10 μM of TLR4 inhibitor TLR4-IN-C34 (C34, MedChemExpress, USA) respectively for 30 min 30 min. Zymosan was used as the positive control at a concentration of 1 mg/mL.

Techniques: Quantitation Assay, Positive Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor cell-released autophagosomes (TRAPs) induce PD-L1-decorated NETs that suppress T-cell function to promote breast cancer pulmonary metastasis

doi: 10.1136/jitc-2024-009082

Figure Lengend Snippet: Tumor cell-released autophagosomes (TRAPs) induce neutrophil extracellular trap (NET) formation via the HMGB1-TLR4-Myd88-ERK/p38 signaling pathway. (A,B) Mouse neutrophils were stimulated with protease K, or ultrasonically treated TRAPs for 3 hours. (A) NETs were examined for DNA (DAPI and SYTOX Green) by fluorescence microscopy, and (B) the levels of dsDNA and myeloperoxidase-DNA (MPO-DNA) complexes in culture supernatants were detected by ELISA. (C) TRAPs pretreated with or without α-HSP27, α-HSP60, α-HSP70, α-HSP90, or α-HMGB1 blocking antibodies, were used to stimulate mouse neutrophils for 3 hours, and then dsDNA and MPO-DNA complexes were detected in culture supernatants. (D,E) Mouse neutrophils were subjected to TRAP Hmgb1 KD or TRAP Hmgb1 NC treatment for 3 hours, (D) NETs were identified by DNA (DAPI and SYTOX Green) by fluorescence microscopy, (E) and the levels of dsDNA and MPO-DNA complexes were detected in culture supernatants. (F) Schematic overview of the mouse tail vein injection with NS, TRAP Hmgb1 KD , or TRAP Hmgb1 NC . (G) Levels of dsDNA and MPO-DNA complexes in the plasma of mice were detected. (H,I) Representative images of immunofluorescence staining for DNA (blue), MPO (green), and H3Cit (red) in lung tissues on day 10. (J,K) Neutrophils pretreated with described inhibitor, or solvent control (DMSO) solutions for 2 hours, were then stimulated with TRAPs for 3 hours. (J) NETs were examined for DNA (DAPI and SYTOX Green) by fluorescence microscopy, and (K) culture supernatants were collected to detect dsDNA and MPO-DNA complexes. (L) Neutrophils from control and TLR4-knockout mice were stimulated with TRAPs for 3 hours, and the levels of dsDNA and MPO-DNA complexes in the culture supernatants were detected. (M) Western blotting analyses of ERK and p38 protein phosphorylation in neutrophils treated with TRAPs at different time points. (N) Neutrophils pretreated with ERK and p38 pathway inhibitors for 1 hour were stimulated with TRAPs for 3 hours, and the levels of dsDNA and MPO-DNA complexes in the culture supernatants were determined. (O) Western blotting analysis of ERK and p38 protein phosphorylation in neutrophils treated with TRAP Hmgb1 KD and TRAP Hmgb1 NC. (P) Neutrophils pretreated with described inhibitors for 2 hours were then stimulated with TRAPs for 1 hour. Western blotting was used to detect the phosphorylation of ERK and P38 proteins. Data (mean±SEM) represent three independent experiments (*p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001; NS, not significant).

Article Snippet: TLR2 inhibitor (TLR2-IN-C29), TLR4 inhibitor (TLR4-IN-C34), ERK inhibitor (SCH772984), and P38 inhibitor (BIRB 796) compounds were purchased from Selleck (Shanghai, China).

Techniques: Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Blocking Assay, Injection, Clinical Proteomics, Immunofluorescence, Staining, Solvent, Control, Knock-Out, Western Blot, Phospho-proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor cell-released autophagosomes (TRAPs) induce PD-L1-decorated NETs that suppress T-cell function to promote breast cancer pulmonary metastasis

doi: 10.1136/jitc-2024-009082

Figure Lengend Snippet: Tumor cell-released autophagosomes (TRAPs) induce neutrophil extracellular trap (NET) formation via the HMGB1-TLR4-Myd88-ERK/p38 signaling pathway. (A,B) Mouse neutrophils were stimulated with protease K, or ultrasonically treated TRAPs for 3 hours. (A) NETs were examined for DNA (DAPI and SYTOX Green) by fluorescence microscopy, and (B) the levels of dsDNA and myeloperoxidase-DNA (MPO-DNA) complexes in culture supernatants were detected by ELISA. (C) TRAPs pretreated with or without α-HSP27, α-HSP60, α-HSP70, α-HSP90, or α-HMGB1 blocking antibodies, were used to stimulate mouse neutrophils for 3 hours, and then dsDNA and MPO-DNA complexes were detected in culture supernatants. (D,E) Mouse neutrophils were subjected to TRAP Hmgb1 KD or TRAP Hmgb1 NC treatment for 3 hours, (D) NETs were identified by DNA (DAPI and SYTOX Green) by fluorescence microscopy, (E) and the levels of dsDNA and MPO-DNA complexes were detected in culture supernatants. (F) Schematic overview of the mouse tail vein injection with NS, TRAP Hmgb1 KD , or TRAP Hmgb1 NC . (G) Levels of dsDNA and MPO-DNA complexes in the plasma of mice were detected. (H,I) Representative images of immunofluorescence staining for DNA (blue), MPO (green), and H3Cit (red) in lung tissues on day 10. (J,K) Neutrophils pretreated with described inhibitor, or solvent control (DMSO) solutions for 2 hours, were then stimulated with TRAPs for 3 hours. (J) NETs were examined for DNA (DAPI and SYTOX Green) by fluorescence microscopy, and (K) culture supernatants were collected to detect dsDNA and MPO-DNA complexes. (L) Neutrophils from control and TLR4-knockout mice were stimulated with TRAPs for 3 hours, and the levels of dsDNA and MPO-DNA complexes in the culture supernatants were detected. (M) Western blotting analyses of ERK and p38 protein phosphorylation in neutrophils treated with TRAPs at different time points. (N) Neutrophils pretreated with ERK and p38 pathway inhibitors for 1 hour were stimulated with TRAPs for 3 hours, and the levels of dsDNA and MPO-DNA complexes in the culture supernatants were determined. (O) Western blotting analysis of ERK and p38 protein phosphorylation in neutrophils treated with TRAP Hmgb1 KD and TRAP Hmgb1 NC. (P) Neutrophils pretreated with described inhibitors for 2 hours were then stimulated with TRAPs for 1 hour. Western blotting was used to detect the phosphorylation of ERK and P38 proteins. Data (mean±SEM) represent three independent experiments (*p< 0.05; **p< 0.01; ***p< 0.001; ****p< 0.0001; NS, not significant).

Article Snippet: TLR2 inhibitor (TLR2-IN-C29), TLR4 inhibitor (TLR4-IN-C34), ERK inhibitor (SCH772984), and P38 inhibitor (BIRB 796) compounds were purchased from Selleck (Shanghai, China).

Techniques: Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Blocking Assay, Injection, Clinical Proteomics, Immunofluorescence, Staining, Solvent, Control, Knock-Out, Western Blot, Phospho-proteomics

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: AHCC inhibited hepatic stellate cells activation by regulation of cytoglobin induction via TLR2-SAPK/JNK pathway and collagen production via TLR4-NF-κβ pathway

doi: 10.1152/ajpgi.00134.2024

Figure Lengend Snippet: AHCC increased cytoglobin production in HSCs via Toll-like receptor 2. A–D : HHSteC cells were treated with AHCC (500 μg/mL) in the presence or absence of TLR2 (50 μM) or TLR4 (5 μM) inhibitor and harvested at the indicated times ( A ) or 48 h after AHCC treatment ( B , C , and D ). The representative Western blotting images for SAPK/JNK signaling are shown. The quantification of Western blot bands was normalized to GAPDH and shown in right panel of each. Data are expressed as means ± SD (WB data are presented from experiments conducted with n = 3). ** P < 0.01 (vs. vehicle). E : HHsteC cells were treated with zymosan (20 or 100 μg/mL) for 48 h. Gene ( left ) and protein ( middle ) of cytoglobin are shown. Right : quantification of Western blot bands was normalized to GAPDH. Data are expressed as means ± SD (WB data are presented from experiments conducted with n = 4). ** P < 0.01, * P < 0.05 (vs. control). AHCC, active hexose correlated compound; Cont, control; HHSteC, human hepatic stellate cells; HSCs, hepatic stellate cells; Inh., inhibitor; NT, no treatment; SAPK/JNK; stress-activated protein kinase/Jun NH 2 -terminal kinase; Veh, vehicle; WB, Western blot.

Article Snippet: TLR2 inhibitor, TLR2-IN-C29 (50 μM) and TLR4 inhibitor, TLR4-IN-C34 (5 μM) were from Selleck Chemicals (Houston, TX).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Berberine protects against gefitinib-induced liver injury by inhibiting the HMGB1/TLR4/NF-κB pathway

doi: 10.3389/fphar.2025.1645634

Figure Lengend Snippet: Gefitinib (GEF) induced upregulation of HMGB1, TLR4, and NF-κB expression, and promotes hepatocyte injury in THLE-2 and THLE-3 cells (n = 3). (A,B) mRNA expression of HMGB1 and TLR4. (C,D) Protein levels of HMGB1, TLR4, and NF-κB (p65 and p-p65). (E,F) ALT and AST levels in the cell supernatant. (G,H) Inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) in the cell supernatant. Cells were treated with GEF (10 μM) for 48 h or co-treated with HMGB1 siRNA, TLR4 inhibitor (TLR4-IN-C34, 20 μM) or NF-κB inhibitor (SC75741, 1 μM). ns, no significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-value < 0.05 was considered statistically significant.

Article Snippet: The specific inhibitors for NF-κB (SC75741) and TLR4 (TLR4-IN-C34) were purchased from Selleck. cn (Shanghai, China).

Techniques: Expressing

Journal: Frontiers in veterinary science

Article Title: Effect of Lactobacillus johnsonii Strain SQ0048 on the TLRs-MyD88/NF-κB Signaling Pathway in Bovine Vaginal Epithelial Cells.

doi: 10.3389/fvets.2021.670949

Figure Lengend Snippet: FIGURE 2 | mRNA expression levels of MyD88 (A,B), IKK (C), NF-κB (D), IL-1β (E), IL-6 (F), TNF-α (G), and IL-10 (H) after treatment with inhibitors in BVECs. Note: (A) TLR4-IN-C34 (5 µm) for 30 min, (B) Sparstolonin B (10 µm) for 120 min; (C) B, PD98,059 (20 µm) for 90 min; (D) Mesalamine (1 mm) for 60 min; (E) Caffeic Acid Phenethyl Ester (20 µm) for 60 min; (F) Caffeic Acid Phenethyl Ester (20 µm) for 60 min; (G) Caffeic Acid Phenethyl Ester (20 µm) for 60 min; (H) Caffeic Acid Phenethyl Ester (20 µm) for 60 min. All data are shown as means ± SD (n = 3). * or # indicating P < 0.05 and ** or ## indicating P < 0.01. *refers to the comparison between cells in the cell control group not exposed to any treatment and other groups exposed to different concentrations of SQ0048 without inhibitor treatment; it also refers to the comparison between the group without inhibitors and the group with inhibitors at the same concentration of SQ0048; # refers to the comparison between the inhibitor control group with inhibitors but not SQ0048 and other groups with inhibitors.

Article Snippet: The names and manufacturers of each factor inhibitor used in this study are as follows: inhibitor of TLR4 is TLR4-IN-C34, co-inhibitor of TLR2 and TLR4 is Sparstolonin B, and inhibitor of NF-κB is Caffeic Acid Phenethyl Ester (Sigma-Aldrich, St. Louis, MO, USA); inhibitor of MEKK1 is B, PD98,059, and inhibitor of IKK is Mesalamine (Selleck Chemicals, Houston, Texas, USA).

Techniques: Expressing, Comparison, Control, Concentration Assay

Journal: Frontiers in veterinary science

Article Title: Effect of Lactobacillus johnsonii Strain SQ0048 on the TLRs-MyD88/NF-κB Signaling Pathway in Bovine Vaginal Epithelial Cells.

doi: 10.3389/fvets.2021.670949

Figure Lengend Snippet: FIGURE 3 | Protein expression levels of MyD88, IKK, and NF-κB after treatment with TLR4-IN-C34 and Sparstolonin (A), B,PD98,059 (B), and Mesalamine (C) in BVECs.

Article Snippet: The names and manufacturers of each factor inhibitor used in this study are as follows: inhibitor of TLR4 is TLR4-IN-C34, co-inhibitor of TLR2 and TLR4 is Sparstolonin B, and inhibitor of NF-κB is Caffeic Acid Phenethyl Ester (Sigma-Aldrich, St. Louis, MO, USA); inhibitor of MEKK1 is B, PD98,059, and inhibitor of IKK is Mesalamine (Selleck Chemicals, Houston, Texas, USA).

Techniques: Expressing

Journal: Frontiers in veterinary science

Article Title: Effect of Lactobacillus johnsonii Strain SQ0048 on the TLRs-MyD88/NF-κB Signaling Pathway in Bovine Vaginal Epithelial Cells.

doi: 10.3389/fvets.2021.670949

Figure Lengend Snippet: FIGURE 4 | Protein expression levels of MyD88 (A,B), IKK (C,D), and NF-κB (E,F) after treatment with various inhibitors in BVECs using grayscale analysis. Note: TLR4-IN-C34 (A) and Sparstolonin B (B); phosphorylated IKKα (C) and non-phosphorylated IKKα (D) in treatment with B,PD98,059; phosphorylated NF-κB (E) and non-phosphorylated NF-κB (F) in the treatment by Mesalamine. All data are shown as means ± SD (n = 3). * or # indicating P < 0.05 and ** or ## indicating P < 0.01. *refers to the comparison between cells in the cell control group not exposed to any treatment and other groups exposed to different concentrations of SQ0048 without inhibitor treatment; it also refers to the comparison between groups without inhibitors and corresponding groups with inhibitors at the same concentration of SQ0048; # refers to the comparison between cells in the inhibitor control group with inhibitors but not SQ0048 and other groups with inhibitors.

Article Snippet: The names and manufacturers of each factor inhibitor used in this study are as follows: inhibitor of TLR4 is TLR4-IN-C34, co-inhibitor of TLR2 and TLR4 is Sparstolonin B, and inhibitor of NF-κB is Caffeic Acid Phenethyl Ester (Sigma-Aldrich, St. Louis, MO, USA); inhibitor of MEKK1 is B, PD98,059, and inhibitor of IKK is Mesalamine (Selleck Chemicals, Houston, Texas, USA).

Techniques: Expressing, Comparison, Control, Concentration Assay