tlr4 inhibitor tlr4i  (Tocris)

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: eNPM binds to TLR4 and the binding increases upon cytokines treatment in HaCaT and HFs. (A) Proximity ligation assay (PLA) of NPM and TLR4 interaction analysis in serum-starved HaCaT cells stimulated with a MIX of cytokines for 8 h (IL-17A, IL-22, TNF-α, and IFN-γ). Lower panels represent zoomed-in images of the original PLA images. Red dots indicate that even under physiological conditions, NPM interacts with TLR4. When a MIX of cytokines is administered, the rate of interaction increases. As a negative control, the IgG specific for each antibody (i.e., normal rabbit IgG used as the control of TLR4 antibody) and normal mouse IgG for NPM antibody (left panel) revealed the specificity of NPM and TLR4 interaction, since no red dots were present in the PLA. Scale bar 10 μm. (B) Bar graph of PLA interactions quantified by the mean number of red dots per cell ( n = 5; * p < 0.05). (C) PLA of NPM and TLR4 interaction analysis in serum-starved HFs derived from healthy subjects and stimulated with a MIX of cytokines for 8 h. Lower panels represent zoomed-in images of the original PLA images. Scale bar 10 μm. (D) Bar graph of PLA interactions quantified by the mean number of red dots per cell ( n = 6; * p < 0.05).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Binding Assay, Proximity Ligation Assay, Negative Control, Control, Derivative Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: rNPM promotes NF-κB translocation to the nucleus and induces inflammation in HaCaT and HFs via TLR4. (A) Representative image of immunofluorescence of NF-kB (red) with an α-p65rel NF-kB antibody, nuclei were counter-stained with DAPI (blue). NFκB translocated in the nucleus in HaCaT treated for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). Mouse IgG isotype control was used as a negative control. Left panels represent zoomed-in images of the original images. Scale bar 5 μm. (B) Representative image of immunofluorescence of NF-kB (red) with an α-p65rel NF-kB antibody, nuclei were counter-stained with DAPI (blue). NF-κB translocated in the nucleus in HFs treated for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). Mouse IgG isotype control was used as a negative control. Left panels represent zoomed-in images of the original images. Scale bar 5 μm. (C) HaCaT were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment ( n = 6; * p < 0.05; ** p < 0.01). (D) HFs were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with rNPM (0.25 μg/ml) compared to untreated cells (w/o NPM). qRT-PCR of COX-2 and IL-6 was significantly reduced by TLR4i upon rNPM treatment ( n = 6; * p < 0.05).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Translocation Assay, Immunofluorescence, Staining, Control, Negative Control, Quantitative RT-PCR

Journal: Frontiers in Cardiovascular Medicine

Article Title: Extracellular Nucleophosmin Is Increased in Psoriasis and Correlates With the Determinants of Cardiovascular Diseases

doi: 10.3389/fcvm.2022.867813

Figure Lengend Snippet: TRL4-based mechanism regulates NPM and miR-200c secretion in response to cytokine mix and miR-200c and NPM physically interact. HaCaT were pre-treated with 100 μM TLR4i for 30 min and then treated or not for 8 h with cytokine MIX (IL-17A, IL-22, TNF-α, and IFN-γ). (A) Media were collected and analyzed by ELISA assay for the presence of eNPM. eNPM increased levels by cytokine MIX were significantly decreased by TLR4i ( n = 5; * p < 0.05; ** p < 0.01). (B) qRT-PCR of miR-200c from the supernatant show that miR-200c increase elicited by cytokine MIX treatment is significantly reduced by TLR4i ( n = 6; ** p < 0.01; *** p < 0.001). (C) HaCaT were treated with cytokine MIX for 8 h then supernatants were collected and treated either with RNase or 200 μM NPM inhibitor (NPMi) or 2 μg/ml RNAse together with 200 μM NPMi for 15 min at 37°C ( n = 6, * p < 0.05; * * p < 0.01).

Article Snippet: Cells were treated with the following drugs administered in a serum-free medium: recombinant NPM (rNPM) was purchased from Abcam (Ab114194) and used 0.25 μg/ml; NPM inhibitor {N1,N2-bis[(3-imino-6-methyl-3H-indol-2-yl)methyl]-N1,N2-bis((6-methyl-1H-benzo[d] imidazol-2-yl)methyl)ethane-1,2-diamine hydrate, NSC348884 Sigma–Aldrich} was dissolved in DMSO and used 200μM, TLR4 inhibitor (TLR4i) is a small peptide [1-methylethyl 2-(acetylamino)-2-deoxy-α-D-glucopyranoside 3,4,6-triacetate, C34 5373/10 Tocris Bioscience] was dissolved in water and used 100 μM; RNAse was used 2 μg/ml.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR