Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Primary peritoneal macrophages from uPAR−/− mice were stimulated with suPAR with or without LPS for 15 min. Then, cells were fixed and stained for TLR4 (Alexa 488, green) and uPAR (Alexa 647, red). DAPI was used as nuclear stain. Scale bar 10 μm. B. Primary WT and uPAR−/− macrophages were stimulated with 100 ng/ml LPS and1μg/ml suPAR for 3 hrs C. Raw 264.7 cells were stimulated with 100 ng/ml LPS, fixed and stained as in A. Scale bar 12.5 μm. D. Raw 264.7 were stimulated with 1μg/ml biotin-LPS for 30 min, then cell lysis was performed. Protein complexes were precipitated using Streptavidin magnetic beads and analyzed by western blotting using anti-murine-uPAR antibody.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Staining, Lysis, Magnetic Beads, Western Blot

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Biotin-LPS binding was assessed in SiCo and uPARsi HK-2 cells as described. B. Duolink proximity ligation assay to assess uPAR/TLR4 and uPAR/CD36 interaction was performed on HK-2 cells stimulated with LPS for 15 min as described in Methods. C. Duolink images were quantified using Particles analysis tool of ImageJ. D. HK-2 cells were stimulated with LPS for 3 hrs after cell pre-treatment with CD36 inhibitor SSO. Expression of IL-6 and IL-8 was assessed by TaqMan RT-PCR.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Binding Assay, Proximity Ligation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Urokinase receptor associates with TLR4 interactome to promote LPS response

doi: 10.1101/2020.06.10.143826

Figure Lengend Snippet: A. Peritoneum of sham and LPS-injected WT mice was fixed and stained for uPAR and TLR4. DAPI used as nuclear stain­ scale bar 100μm. B. Expression of TNFα, MCP-1, IL-6 and IL-1 0 was assessed in mouse blood plasma before and 20 h after CLP surgery using Cytometric Beads Array. C: IL-6/IL-10 ratio in CLP mice 20 hrs after surgery.

Article Snippet: Unconjugated and Alexa 647-conjugated mouse uPAR antibody were from R&D systems (MAB531 and FAB531R, respectively); TLR4 antibody (MAB27591) was from SrnD Systems; LPS (L2887) was from Sigma, Biotin-LPS and PAM3CSK4 were from Invivogen; Soluble mouse uPAR was from CinoBiologicals.

Techniques: Injection, Staining, Expressing, Clinical Proteomics

Journal: The Journal of pharmacy and pharmacology

Article Title: Mangiferin attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting TLR4/p65 and TGF-β1/Smad2/3 pathway.

doi: 10.1111/jphp.13077

Figure Lengend Snippet: Figure 5 The effect of mangiferin on the expression of TLR4 and p-P65 among different experimental groups. (a) Representative images of TLR4 immunofluorescence staining in lung sections; (c) representative images of p-P65 immunofluorescence staining of lung sections; and (b and d) the relative quantitative expression analysis of TLR4 and p-P65. Data are shown as mean SD. n = 6 in each group. #P < 0.01 vs control group; *P < 0.01 vs bleomycin model group.

Article Snippet: Anti-mouse TLR4, p-P65, TGF-b1, a-SMA, E-Cadherin, MMP-9, p-Smad2/3 and Smad2/3 primary antibodies were purchased from R&D Corporation (R&D Systems, Minnneapolis, MN, USA).

Techniques: Expressing, Staining, Control

Journal: EMBO Reports

Article Title: Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity

doi: 10.15252/embr.202051280

Figure Lengend Snippet: Activity of an NF‐κB reporter relative to vehicle control in human embryonic kidney cells that express TLR4 (hTLR4) or an isogenic control cell line that does not express TLR4 (null2) and were stimulated with vehicle (veh), 1 ng/ml LPS, 400 µM nickel chloride, or 25, 50, or 100 µM platinum(II) chloride, and platinum(IV) chloride ( n = 3 independent biological replicates). As per panel (A), secreted IL‐8 was monitored as a metric of TLR4 activation upon stimulation with 50 pg/ml LPS, 200 µM nickel chloride, 100 µM platinum(II) chloride, or 100 µM platinum(IV) chloride ( n = 4 independent biological replicates). IL‐8 secretion in HEK‐hTLR4 and HEK‐null2 cells following treatment with cisplatin at the indicated concentrations ( n = 3 independent biological replicates). IL‐8 secretion in HEK‐hTLR4 cells pre‐treated with 4 µ M TAK242 (TLR4 inhibitor) or vehicle, and subsequent treatment with 50 pg/ml LPS, 200 µM nickel chloride, or 25 µM cisplatin ( n = 3 independent biological replicates). Mock cells were not subject to pre‐treatment prior to agonist addition. Data Information: For all panels, actual individual data from each experiment are plotted as box (25 th and 75 th percentile borders; median central band) with Tukey whiskers. Statistical analyses were assessed by 2‐way ANOVA: in (A) hTLR4 compared to null2 cells; in (B) agonist treatment compared to non‐treated ( nil ); in (C) comparisons between successive concentrations; and in (D) comparisons between vehicle and TAK‐242 treatments. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (Dunnett’s test, A, B, D; Tukey test, C). Source data are available online for this figure.

Article Snippet: HEI‐OC1 cells were transfected with a mouse Tlr4 expression clone (OriGene; MR210887) to test for complementation of the Tlr4 deletion strain.

Techniques: Activity Assay, Control, Activation Assay

Journal: EMBO Reports

Article Title: Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity

doi: 10.15252/embr.202051280

Figure Lengend Snippet: IL‐8 secretion in HEK cells stably expressing hTLR4 but not MD‐2 (HEK‐isoTLR4), transfected with empty vector (EV) or MD‐2 and left untreated ( nil ) or treated with 1 ng/ml LPS, 200 µM nickel chloride, 2 µg/ml HMGB1, 100 µM platinum(II) chloride, 100 µM platinum(IV) chloride, or 25 µM cisplatin ( n = 3 or 4 independent biological replicates). Fold IL‐8 secreted (relative to nil treatment) in MD‐2‐deficient HeLa cells treated with 10 or 100 ng/ml LPS or 25 µM cisplatin ( n = 4 independent biological replicates). IL‐8 secretion in HeLa cells transfected with non‐targeting (siNT) or TLR4 ‐targeting (si TLR4 ) siRNA and left untreated ( nil ), or treated with 30 µM cisplatin ( n = 3 independent biological replicates). Mock cells were not subject to siRNA treatment prior. Data Information: Actual individual data are plotted as box (25 th and 75 th percentile borders; median central band) with Tukey whiskers (A, C) or mean and standard deviation (B). Statistical analyses were determined in comparison to nil treatments using 2‐way (A, C) or one‐way (B) ANOVA. ns, not significant; * P < 0.05; **** P < 0.0001 (Dunnett’s test). Source data are available online for this figure.

Article Snippet: HEI‐OC1 cells were transfected with a mouse Tlr4 expression clone (OriGene; MR210887) to test for complementation of the Tlr4 deletion strain.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Standard Deviation, Comparison

Journal: EMBO Reports

Article Title: Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity

doi: 10.15252/embr.202051280

Figure Lengend Snippet: TLR4 expression levels (relative to nil treatment) in HeLa cells transfected with non‐targeting (siNT) or TLR4 ‐targeting (si TLR4 ) siRNA molecules and treated with 30 µM cisplatin. Data information: Actual individual data from 2 independent experiments are plotted with mean and standard deviation indicated. Source data are available online for this figure.

Article Snippet: HEI‐OC1 cells were transfected with a mouse Tlr4 expression clone (OriGene; MR210887) to test for complementation of the Tlr4 deletion strain.

Techniques: Expressing, Transfection, Standard Deviation

Journal: EMBO Reports

Article Title: Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity

doi: 10.15252/embr.202051280

Figure Lengend Snippet: Comparison of genomic DNA at the Tlr4 locus from Tlr4 −/− HEI‐OC1 and wild‐type cells. Sequences from the Tlr4 −/− cell line contained a single nucleotide insertion or four nucleotide deletion and summarized below. Anti‐TLR4 staining in Tlr4 −/− and control HEI‐OC1 cells. Bars (lower right) are 50 µm. Flow cytometric analysis of conjugated LPS internalization in Tlr4 −/− and control HEI‐OC1 cells ( n = 4 independent biological replicates). IL‐6 secretion in Tlr4 −/− and control HEI‐OC1 cells transfected with empty vector (EV), Tlr4 (p Tlr4 ), or left untransfected (−) and subsequently treated with 100 ng/ml LPS ( n = 4 independent biological replicates). Inset , fold induction of IL‐6 secretion was determined relative to the untransfected cells treated with LPS. Data information: In (C and D), data are presented as mean and standard deviation. Statistical comparisons were assessed by 2‐way ANOVA (D). ** P < 0.01; **** P < 0.0001 (unpaired Student’s t ‐test, C; Bonferroni test, D). Source data are available online for this figure.

Article Snippet: HEI‐OC1 cells were transfected with a mouse Tlr4 expression clone (OriGene; MR210887) to test for complementation of the Tlr4 deletion strain.

Techniques: Comparison, Staining, Control, Transfection, Plasmid Preparation, Standard Deviation

Journal: EMBO Reports

Article Title: Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity

doi: 10.15252/embr.202051280

Figure Lengend Snippet: A–D HEI‐OC1 cells containing a Tlr4 deletion ( Tlr4 −/− ) were compared to HEI‐OC1 non‐targeting (NT) control cells and assessed for cell viability (A), Annexin V/propidium iodide staining (B), ROS generation (C), and IL‐6 secretion (D) following cisplatin treatment at the indicated concentrations ( n = 3 independent biological replicates). Data Information: In all panels, data are presented as mean and standard deviation. Statistical comparisons to NT at the same cisplatin concentration were assessed by 2‐way (A, B) or one‐way (C, D) ANOVA. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (Bonferroni test). Source data are available online for this figure.

Article Snippet: HEI‐OC1 cells were transfected with a mouse Tlr4 expression clone (OriGene; MR210887) to test for complementation of the Tlr4 deletion strain.

Techniques: Control, Staining, Standard Deviation, Concentration Assay

Journal: EMBO Reports

Article Title: Toll‐like receptor 4 is activated by platinum and contributes to cisplatin‐induced ototoxicity

doi: 10.15252/embr.202051280

Figure Lengend Snippet: IL‐6 secretion in HEI‐OC1 cells treated with 100 pg/ml LPS or 20 µM cisplatin ( n = 3 or 4 independent biological replicates). IL‐6 secretion in TLR4 −/− cells transfected with empty vector (EV) or mouse Tlr4 (m Tlr4 ) following treatment with 20 µM cisplatin ( n = 6 independent biological replicates). Il6 and Tlr4 transcript levels in HEI‐OC1 cells following treatment with 20 µM cisplatin for the indicated times ( n = 3 independent biological replicates). Data information: In all panels, data are presented as mean and standard deviation. Statistical comparisons to 0 h time point were assessed by 2‐way ANOVA. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 (Dunnett’s test). Source data are available online for this figure.

Article Snippet: HEI‐OC1 cells were transfected with a mouse Tlr4 expression clone (OriGene; MR210887) to test for complementation of the Tlr4 deletion strain.

Techniques: Transfection, Plasmid Preparation, Standard Deviation

Journal: bioRxiv

Article Title: High-integrity nanoemulsions formulation of resiquimod (R848) enhances stability and delivery for triple negative breast cancer immunotherapy

doi: 10.1101/2025.04.04.647265

Figure Lengend Snippet: ELISA data was obtained from 4T1 breast cancer cells treated with NE formulations, compared to LPS-treated and untreated cells.

Article Snippet: TLR4 levels were determined using the Mouse TLR4 ELISA Kit (Elabscience ® , Cat. No. E-EL-M2417), while TLR7 was measured with the Mouse Toll-like Receptor 7 ELISA Kit (MyBioSource, Cat. No. MBS2533523).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A novel reporter gene assay for pyrogen detection

doi: 10.1101/613190

Figure Lengend Snippet: Expression of TLR2 (green line), TLR4 (orange line), and TLR6 (purple line) on RAW 264.7 cells (a) and binding curves of the corresponding antibodies (Ab, concentrations from 10 μg/ml to 0.005 μg/ml, at 1:3 dilutions) to TLR2, TLR4, and TLR6 on RAW 264.7 cells (b) were analysed by FACS. Rat IgG2a AF488 (blue shadow or line) was used as an isotype control.

Article Snippet: Then, 50 μl of 2 μg/ml fluorescence-labelled antibodies was added for the detection of TLR2 (R&D, Cat # FAB1530G), TLR4 (R&D, Cat # FAB27591G), and TLR6 (R&D, Cat # FAB1533G); rat IgG2a Alexa Fluor (AF) 488-conjugated antibody was used as an isotype control (R&D, Cat # IC006G).

Techniques: Expressing, Binding Assay

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 2. Proximal stiffening induces proinflammatory response and upregulates TLR2 expression in downstream bovine PAECs. (A-B) High pulsatility flow (HPF), due to the use of a “stiff” tube upstream to cell culture, upregulated proinflammatory molecule mRNA (ICAM-1, VCAM-1, MCP-1 and E-selectin) and protein (MCP-1) in healthy PAECs, compared to low pulsatility flow (LPF) or to static conditions. (C-D) The mRNA and protein expression of TLR2 but not TLR4 in PAECs was highly upregulated by HPF. *: p<0.05 versus Static, †: p<0.05 versus LPF. https://doi.org/10.1371/journal.pone.0102195.g002

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Expressing, Cell Culture

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 3. Pharmacological or siRNA inhibition of TLR2 results in suppression of PAEC proinflammatory responses caused by stiffening-induced HPF. (A, C) At the mRNA level, TLR2/4 inhibitor OxPAPC or TLR2 siRNA but not TLR4 inhibitor CLI-095, decreased PAEC expression of ICAM-1, VCAM-1, MCP-1 and E-selectin mRNAs under HPF; TLR4 siRNA decreased ICAM-1 and E-selectin but not VCAM-1 and MCP-1 mRNAs. “*”: p<0.05 versus LPF, “†”: p<0.05 versus HPF. (B, D) At the protein level, the MCP-1 expression in PAECs exposed to HPF was inhibited by OxPAPC or TLR2 siRNA treatment but not CLI-095 or TLR4 siRNA. The black line in the blot images (D, right) shows separated lanes obtained on the same gel. https://doi.org/10.1371/journal.pone.0102195.g003

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Inhibition, Expressing

Journal: PloS one

Article Title: Stiffening-induced high pulsatility flow activates endothelial inflammation via a TLR2/NF-κB pathway.

doi: 10.1371/journal.pone.0102195

Figure Lengend Snippet: Figure 6. Enhanced TLR and MCP-1 expression in the distal pulmonary artery endothelium in vivo. (A) PAECs from calves with hypoxia-induced pulmonary hypertension (PH) show elevated expression of TLR2 and TLR4, compared to control (CO). *:p<0.05. (B) Both immunostaining and western blotting results show elevated MCP-1 expression by PH-ECs compared to CO-ECs from calves. “PA” indicates the lumen of a pulmonary artery. *:p<0.05. (C) Enhanced TLR2 expression in the pulmonary arterial endothelium of human with pulmonary arterial hypertension (PAH). Cryosections of human intra-lobar pulmonary arteries were immunostained with TLR2 (red fluorescence) and counterstained with DAPI (cell nuclei, blue). Elastic lamellae showed green auto-fluorescence. https://doi.org/10.1371/journal.pone.0102195.g006

Article Snippet: Antibodies used here include: rabbit polyclonal antibody against bovine MCP-1 (1∶500 dilution; Kingfisher Biotech, St. Paul, MN), rabbit polyclonal antibody against TLR2 (1∶100 dilution; Bioss Inc, Woburn MA), mouse monoclonal antibody against TLR4 (1∶100 dilution; Acris Antibodies Inc, San Diego, CA) and mouse monoclonal antibody against GAPDH (1∶20,000 dilution; Sigma Inc, Saint Louis, MO).

Techniques: Expressing, In Vivo, Control, Immunostaining, Western Blot