tlr3 antagonist  (Tocris)

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: Primary antibodies used for immunofluorescence staining.

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Immunofluorescence, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: EEA1 decreases and LAMP1 expression increases depending on poly(I:C) stimulation time of C8-D1A murine astrocytes, while increasing TLR3 ligand concentration augments the expression of LAMP1. Representative western blots of EEA1, LAMP1, and Rab7 expression in C8-D1A cells treated with poly(I:C) at concentration 10 μg/ml (A) , with poly(I:C)/LyoVec at concentration 1 μg/ml (C) , or with LyoVec at concentration 1 μg/ml (E) , and lysed at various times following stimulation (0, 30 min, 1, 2, 4, 8, and 24 h). EEA1, LAMP1, and Rab7 expression was also analyzed in cells treated with different concentrations of poly(I:C) (0; 0,1; 1; 2; 5; 10 μg/ml) (B) , poly(I:C)/LyoVec (0; 0,1; 1; 2; 5 μg/ml) (D) , or LyoVec (0; 0,1; 1; 2; 5 μg/ml) (F) , and lysed 24 h following stimulation. GAPDH levels are shown to evaluate equal gel loading. The densitometry analysis of EEA1, LAMP1, and Rab7 was performed in cells treated with 10 μg/ml poly(I:C) for indicated time points (A) ; indicated poly(I:C) concentrations for 24 h (B) ; 1 μg/ml poly(I:C)/LyoVec for indicated time points (C) ; indicated poly(I:C)/LyoVec concentrations for 24 h (D) ; 1 μg/ml LyoVec for indicated time points (E) ; or indicated LyoVec concentrations for 24 h (F) . Protein expression levels were normalized to GAPDH and presented as relative expression (mean ± SD). The GFP/SEAP reporter was efficiently delivered into C8-D1A cells when LyoVec was used as a transfectant (G) . Statistical comparisons were performed between untreated and poly(I:C)- or poly(I:C)/LyoVec- or LyoVec-treated astrocytes (two sample Student’s t -test; * P ≤ 0.05; ** P ≤ 0.01). Cells transfected with LyoVec alone were used as a control to disengage the contribution of the membrane solubilization as well as interaction of the cationic detergent with cellular compartments. Data were obtained from three independent experiments.

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Expressing, Concentration Assay, Western Blot, Transfection, Control, Membrane

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: TLR3 is present in EEA1-labeled endosomes at least up to 1 h following poly(I:C) stimulation of murine astrocytes. C8-D1A cells were not treated or treated with poly(I:C) for 1, 8, 12, 20, 30 min, 1, 2, 8, and 24 h. The astrocytes were then fixed and stained with specific antibodies for EEA1 (green) and TLR3 (red). Magn. – magnification of the selection marked with a white frame on the merged image (merge). White arrows indicate the TLR3 and EEA1 co-localization sites. Experiments were performed at least three times and only representative images are shown. Scale bar: 10 μm. Indirect PLA was performed to study the co-localization of EEA1 and TLR3 in not treated cells or in cells following 1 h of poly(I:C) stimulation. Positive PLA (red dots) was observed in cells stimulated with poly(I:C) for 1 h (see ).

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Labeling, Staining, Selection

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: Astrocytic TLR3 is constantly present in late endosomes, however, TLR3 and Rab7 co-localization increases with the time of poly(I:C) stimulation. C8-D1A murine astrocytes were not stimulated or stimulated with poly(I:C) for 1, 8, 12, 20, 30 min, 2, 4, 8, and 24 h. Co-localization of Rab7 (green) and TLR3 (red) occurred in vesicular structures, which can be seen on merged images (merge). All merged pictures have been enlarged in the white frame area where the co-localization of Rab7 and TLR3 can be seen (magn.). White arrows indicate co-localization sites. Experiments were performed at least three times and only representative images are shown. Scale bar: 10 μm. Indirect PLA was performed to investigate the co-localization of Rab7 and TLR3 in untreated astrocytes or in astrocytes treated with poly(I:C) for 24 h. Positive PLA (red dots) was observed in cells stimulated with poly(I:C) for 24 h (see ).

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: TLR3 is abundantly translocated to LAMP1-labeled endosomes after poly(I: C) stimulation of C8-D1A cells. Mouse astrocytes were not treated or treated with poly(I:C) for 1, 8, 12, 20, 30 min, 2, 8, and 24 h. The cells were then fixed and labeled with anti-LAMP1 (green) and anti-TLR3 (red) antibodies. All merged images (merge) have been enlarged in the area where the co-localization of LAMP1 and TLR3 can be seen (magn.) and white arrows indicate co-localization sites. Experiments were performed at least three times and the representative images are shown. Scale bar: 10 μm. To explore the co-localization of LAMP1 and TLR3 in untreated cells or in cells following 24 h of poly(I:C) stimulation, indirect PLA was performed. Positive PLA (red dots) was observed in cells stimulated with poly(I:C) for 24 h (see ).

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: TLR3 interacts with poly(I:C) from the first minute following stimulation of C8-D1A cells with the TLR3 ligand. C8-D1A cells were not stimulated or stimulated with poly(I:C) for 1, 8, 12, 20, 30 min, 1, 2, 4, 8, and 24 h. The astrocytes were then fixed and labeled with anti-dsRNA (green) and anti-TLR3 (red) antibodies. All merged images (merge) have been enlarged in the area where the co-localization of poly(I:C) and TLR3 can be seen (magn.). White arrows indicate the TLR3 and poly(I:C) co-localization sites. Experiments were performed at least three times and the representative images are shown. Scale bar: 10 μm.

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Labeling

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: Astrocytic TLR3 is not present in early endosomes, late endosomes or lysosomes following pretreatment with the TLR3 inhibitor, CU CPT 4a, and subsequent poly(I:C) stimulation. C8-D1A murine astrocytes were stimulated with poly(I:C) for 1 or 24 h. Co-localization of EEA1 (A) , Rab7 (B) or LAMP1 (C) (green) and TLR3 (red) occurred in vesicular structures, however, no interaction of the studied proteins was observed following treatment of cells with CU CPT 4a (20 μM) for 1 h before the addition of poly(I:C). Merged images (merge) have been enlarged in the area where the co-localization of EEA1 and TLR3 (A) , Rab7 and TLR3 (B) , and LAMP1 and TLR3 (C) can be seen (magn.) and white arrows indicate co-localization sites. (D) ELISA assay result demonstrates that CU CPT 4a significantly inhibits TLR3 and subsequently IFN-β secretion following stimulation with 10 μg/ml poly(I:C) in C8-D1A cells. Statistical comparisons were performed between untreated and poly(I:C)- or CU CPT 4a and poly(I:C)-treated astrocytes (two sample Student’s t -test; * P ≤ 0.05; ** P ≤ 0.01). Experiments were performed at least three times and only representative images are shown. Scale bar: 10 μm.

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: TLR3 is constitutively expressed on the surface of C8-D1A murine astrocytes and is enhanced following stimulation with the TLR3 agonist. Representative cytograms show surface TLR3 protein expression on the cells: not treated with poly(I:C); or treated for 15, 30 min, 1, 2, 4, 24 h; isotype control; intracellular staining control (A) . The isotype control serves as the negative control and functions to validate the result that TLR3 present on the surface of astrocytes is not an artifact. The intracellular staining control serves as the positive control to validate the specific anti-TLR3 antibodies used in the experiment by staining the intracellular TLR3, following permeabilization of the cells. P4 – astrocyte population where TLR3 occurs on the surface. Numbers in the dot plots represent the mean percentage ± SD of surface TLR3(+) cells in the three independent experiments. The cytograms show green fluorescence on the x -axis and red fluorescence on the y -axis. (B) Graph showing the occurrence of TLR3 on the cell surface at various times following poly(I:C) treatment (% of all cells). Statistical comparisons were performed between untreated and poly(I:C)-treated astrocytes (two sample Student’s t -test; * P ≤ 0.05; ** P ≤ 0.01). Experiments were performed three times, and each dot plot represents mean ± SD. The graph shows the time of astrocytic stimulation with poly(I:C) on the x -axis, and the percentage of cells where TLR3 occurred on the surface on the y -axis.

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Expressing, Control, Staining, Negative Control, Positive Control, Fluorescence

Journal: Frontiers in Cellular Neuroscience

Article Title: Participation of Endosomes in Toll-Like Receptor 3 Transportation Pathway in Murine Astrocytes

doi: 10.3389/fncel.2020.544612

Figure Lengend Snippet: Viruses that induce TLR3 signaling leading to generation of a protective response.

Article Snippet: To exclude contribution of the non-TLR3-mediated signaling pathways, we used TLR3 antagonist, N -[(3-chloro-6-fluorobenzo[b]thien-2-yl)carbonyl]-d-phenylalanine (CU CPT 4a; Tocris Bioscience).

Techniques: Virus

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

doi: 10.1210/jc.2019-00273

Figure Lengend Snippet: Figure 3. HT exosomes bind to and upregulate TLR2/TLR3. DCs were cocultured with HT exosomes or HC exosomes for 24 hours. (a) Immunofluorescence analysis showed that DCs (blue color, marked by DAPI) were colocalized with HT exosomes or HC exosomes (green color, marked by PKH-67) after coculture for 24 hours. TLR2 and TLR3 were marked by Alexa Fluor 594 (red color) (magnification 3400, confocal laser-scanning microscopy). PBMCs were cocultured with HT exosomes or HC exosomes for 24 hours. (b) Representative patterns of CD11c1TLR21, CD11c1TLR31, and CD11c1TLR41 cells as analyzed by flow cytometry. (c) Frequency of CD11c1TLR21, CD11c1TLR31, and CD11c1TLR41 cells in the HT exosomes and HC exosomes groups (n 5 20 per group). The PBS group was used as a negative control (n 5 4). Statistical analyses of multiple groups were performed by Kruskal-Wallis H-test or one-way ANOVA. *P , 0.05; **P , 0.01.

Article Snippet: To perform the inhibition test, according to the literature and the results of our preliminary experiments, DCs were preincubated with the TLR2 antagonist PAb hTLR2 at a concentration of 3 mg/mL (Invitrogen, Carlsbad, CA) or with the TLR3 antagonist CU CPT 4a at a concentration of 30 mg/mL (Tocris Bioscience, Ellisville, MOUK) for 3 hours and then stimulated with 600 mg HT exosomes.

Techniques: Immunofluorescence, Confocal Laser Scanning Microscopy, Flow Cytometry, Negative Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

doi: 10.1210/jc.2019-00273

Figure Lengend Snippet: Figure 4. HT exosomes activate the NF-kB signaling pathway in DCs, upregulate the expression of costimulatory molecules, and increase the release of IL-6. DCs were cocultured with HT exosomes or HC exosomes for 24 hours. To perform the inhibition experiments, DCs were pretreated with TLR2/3 inhibitors for 3 hours and then incubated with HT exosomes for 24 hours. (a) Representative stripe images and protein expression of MyD-88, TRIF, and p-P65 in the HC exosomes, HT exosomes (n 5 17 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 8 to 10 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 5). TLR2 inhibitor and TLR3 inhibitor groups were used as positive controls in the inhibition experiments (n 5 8 to 10 per group). The relative protein expression level was corrected to the GAPDH expression level. (b) Expression of IL-6 in the supernatants of the HC exosomes, HT exosomes (n 5 14 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 8 to 13 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 5). TLR2 inhibitor and TLR3 inhibitor groups were used as positive controls in the inhibition experiments (n 5 5). (c) Expression of the costimulatory molecules CD40, CD80, and CD83 on CD11c1DCs in the HC exosomes (n 5 11), HT exosomes (n 5 10), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 6 to 8 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 5). TLR2 inhibitor and TLR3 inhibitor groups were used as positive controls in the inhibition experiments (n 5 6 to 4). Statistical analyses of multiple groups were performed by Kruskal-Wallis H-test or one-way ANOVA. *P , 0.05; **P , 0.01.

Article Snippet: To perform the inhibition test, according to the literature and the results of our preliminary experiments, DCs were preincubated with the TLR2 antagonist PAb hTLR2 at a concentration of 3 mg/mL (Invitrogen, Carlsbad, CA) or with the TLR3 antagonist CU CPT 4a at a concentration of 30 mg/mL (Tocris Bioscience, Ellisville, MOUK) for 3 hours and then stimulated with 600 mg HT exosomes.

Techniques: Expressing, Inhibition, Incubation, Negative Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

doi: 10.1210/jc.2019-00273

Figure Lengend Snippet: Figure 5. HT exosomes modulate the differentiation of Th1, Th17, and Treg cells and the secretion of cytokines. PBMCs were incubated with HT exosomes or HC exosomes for 1 to 4 days. To perform the inhibition experiments, PBMCs were pretreated for 3 hours with TLR2 or TLR3 inhibitors and then incubated with HT exosomes for 1 to 4 days. (a) Representative pictures of CD41IFN-g1Th1, CD41IL-17A1Th17A, and CD41CD251Foxp31 Treg cells as analyzed by flow cytometry in the inhibition experiments. (b) Frequency of CD41IFN-g1Th1, CD41IL- 17A1Th17A, and CD41CD251Foxp31 Treg cells in the HC exosomes, HT exosomes (n 5 30 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 10 to 12 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 3). TLR2 inhibitor and TLR3 inhibitor groups were used as a positive control in the inhibition experiments (n 5 5). (c) Levels of IFN-g, IL-17A, and IL-10 in the cell supernatants as determined by ELISA in the HC exosomes, HT exosomes (n 5 18 to 23 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 12 to 20 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 3). TLR2 inhibitor and TLR3 inhibitor groups were used as a positive control in the inhibition experiments (n 5 5). HT-exo 1 TLR2 inhibitor: HT exosomes 1 TLR2 inhibitor; HT-exo 1 TLR3 inhibitor: HT exosomes 1 TLR3 inhibitor. Statistical analyses of multiple groups were performed by Kruskal-Wallis H-test or one-way ANOVA. *P , 0.05; **P , 0.01.

Article Snippet: To perform the inhibition test, according to the literature and the results of our preliminary experiments, DCs were preincubated with the TLR2 antagonist PAb hTLR2 at a concentration of 3 mg/mL (Invitrogen, Carlsbad, CA) or with the TLR3 antagonist CU CPT 4a at a concentration of 30 mg/mL (Tocris Bioscience, Ellisville, MOUK) for 3 hours and then stimulated with 600 mg HT exosomes.

Techniques: Incubation, Inhibition, Flow Cytometry, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay