thp1 dual ko tlr4 md  (InvivoGen)

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Cell Culture, Refractive Index, IF-cells, Concentration Assay, Control, Incubation, Generated

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: Pharmacological parameter of LPS induced DMR at six selected time points in HEK293 TLR4/MD-2/CD14 reporter cells

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques:

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Suspension, Two Tailed Test

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Control, Incubation

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Isolation

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Immunofluorescence, Microscopy, Transfection, Construct

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a DMR principle: cultured cells are grown in 384 well microplates equipped with a resonant waveguide grating biosensor within the bottom of each well. A specific broadband light source illuminates the lower surface of the biosensor. The illumination generates an energy field within the DMR detection zone, and its strength diminishes exponentially with the distance from the sensor. Under baseline conditions (left) cells are in close proximity to the biosensor, and the refractive index of these cells determines the reflected wavelength, which is subsequently measured. If cells undergo morphological changes (middle), the refractive indices above the sensor can either increase or decrease. Consequently, the reflected wavelength becomes shorter or longer, respectively. The shift in the measured wavelength is plotted against the recording time (right). Schematic illustration adapted from ref. ) b Schematic representation of TLR4 signaling and contact point of TAK-242. c , d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli , S. minnesota and R. sphaeroides (1000 ng/ml). Dashed lines represent the six time points that were used to generate concentration-effect curves ( h ). e HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with 50 µM of the TLR4 antagonist TAK-242 or ( f ) HEK293 control reporter cells lacking TLR4 (Null2) were stimulated with LPS E . coli . (1000 ng/ml). g HEK293 TLR4/MD-2/CD14 reporter cells were incubated with TAK-242 (50 µM). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. h Sigmoidal concentration effect curves resulting from DMR traces ( c ) of n biologically independent experiments ( h : n = 4 biological replicates except LPS E . coli 50 min n = 3 (Mean ± SEM)). Concentration-effect curves of DMR data were generated by the response at six different time points. Calculated pharmacological parameters of the concentration-effect curves are depicted in Table . Source data are provided as a Source Data file. ( a ) and ( b ) was created in BioRender. Weindl, G. (2024) a : BioRender.com/k68r667 b : BioRender.com/j94y620.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Cell Culture, Refractive Index, IF-cells, Concentration Assay, Control, Incubation, Generated

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: Pharmacological parameter of LPS induced DMR at six selected time points in HEK293 TLR4/MD-2/CD14 reporter cells

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques:

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a HEK293 TLR4/MD-2/CD14 reporter cells in suspension were stimulated with the indicated concentrations (ng/ml) of LPS from E. col i with or without 50 µM of the TLR4-antagonist TAK-242. HEK293 TLR4/MD-2/CD14 reporter cells were preincubated with the indicated concentrations of actin and microtubule inhibitors ( b ) cytochalasin B, ( c ) latrunculin A or ( d ) nocodazole stimulated with LPS E. coli (1000 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. ( a – d , right panels) Values at 250 min are presented as mean + SEM and are normalized to LPS E. coli (1000 ng/ml) ( n = 3 biologically independent experiments). Two-tailed Student's t test ( a ) and one-way analysis of variance (ANOVA, Tukey’s post-test) ( b – d ). Source data are provided as a Source Data file.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Suspension, Two Tailed Test

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: THP-1 monocytes ( a ) or macrophages ( b ) were stimulated with increasing concentrations (ng/ml) of LPS E.coli and LPS S. minnesota . THP-1 KO-TLR4 monocytes ( c ) or macrophages ( d ) were stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . THP1-Dual TLR4/MD-2/CD14 (THP-1 Dual) ( e ) or THP1-Dual KO-TLR4/MD-2/CD14 (THP-1 Dual TLR4-KO) ( f ) cells stimulated with increasing concentrations (ng/ml) of LPS E. coli and LPS S. minnesota . Heatmap of the top 100 significant up- or downregulated genes identified in HEK293 TLR4/MD-2/CD14 cells ( g ) or THP-1 macrophages ( h ) treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. The global transcriptional response induced by the LPS chemotypes showed no significant differences. n = 2 biologically independent experiments. Venn diagram for HEK293 TLR4/MD-2/CD14 cells ( i ) and THP-1 macrophages ( j ) indicating the number of significant (FDR < 0.05) differentially expressed genes and the overlap between each set of genes treated with buffer only (control), LPS E. coli or LPS S. minnesota , after 3 h incubation. k , l THP1 monocytes (k) or macrophages (l) were stimulated with increasing concentrations (ng/ml) Pam 3 CSK 4 or Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Control, Incubation

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Primary monocytes isolated from PBMCs were stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S. minnesota . b Primary monocytes isolated from PBMCs were preincubated with 50 µM of the TLR4 antagonist TAK-242 and stimulated with the indicated concentrations (ng/ml) of LPS from E. coli or S . minnesota . c Primary monocytes isolated from PBMCs stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 and Pam 2 CSK 4 . Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments and donors. Source data are provided as a Source Data file.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Isolation

Journal: Nature Communications

Article Title: Label-free biosensor assay decodes the dynamics of Toll-like receptor signaling

doi: 10.1038/s41467-024-53770-9

Figure Lengend Snippet: a Schematic representation of TLR4 signaling, ligands used and contact point of the MyD88 inhibitor ST2825. HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) ( b ) or S. minnesota (100 ng/ml) or preincubated with 10 µM of the MyD88 inhibitor ST2825. c Immunofluorescence microscopy for localization experiments of MyD88 (green) in transfected HEK293 KO-MyD88 cells before and after stimulation with LPS E. coli and LPS S. minnesota (100 ng/ml) for 5 min, 15 min or 45 min. Cells are transfected with 500 ng MyD88-Venus construct and counterstained with the nuclear probe Hoechst (blue). Scale bars, 5 µm. Images are representative of three biologically independent experiments. d HEK293 TLR4/MD-2/CD14 reporter cells were stimulated with LPS from E. coli (100 ng/ml) or S. minnesota (100 ng/ml). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. Source data are provided as a Source Data file. ( a ) was created in BioRender. Weindl, G. (2024) BioRender.com/x63i940.

Article Snippet: THP1-Dual TLR4/MD-2/CD14 (#thpd-mctlr4) and THP1-Dual KO-TLR4/MD-2/CD14 (#thpd-mckotlr4) cells (InvivoGen, Toulouse, France) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), HEPES (25 mM), normocin (100 µg/ml) and the selective antibiotics blasticidin (10 µg/ml) and zeocin (100 µg/ml) following the manufacturer’s instructions.

Techniques: Immunofluorescence, Microscopy, Transfection, Construct

Journal: Frontiers in Immunology

Article Title: Aged Gut Microbiota Contributes to Systemical Inflammaging after Transfer to Germ-Free Mice

doi: 10.3389/fimmu.2017.01385

Figure Lengend Snippet: Transfer of old microbiota enhances inflammatory bacterial components in serum. Human Embryonic Kidney 293 cells transfected with mouse toll-like receptor (TLR)2/CD14 (A) or mouse TLR4/MD-2/CD14 (B) were stimulated with 2.5% serum from young or old conventional (conv) mice ( n = 10), germ-free (GF) recipient mice of young or old microbiota ( n = 10), and GF control mice ( n = 5). Activation of these receptors was measured with a secreted embryonic alkaline phosphatase reporter coupled to the NF-kB/AP-1 promoter. All data are expressed as means. ** P < 0.01.

Article Snippet: Human Embryonic Kidney 293 cells stably transfected with mouse TLR2/CD14 or TLR4/MD-2/CD14 and the secreted embryonic alkaline phosphatase reporter coupled to the NF-kB/AP-1 promoter were purchased from Invivogen (San Diego, CA, USA).

Techniques: Transfection, Activation Assay

Journal: Nucleic Acids Research

Article Title: Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9

doi: 10.1093/nar/gkt078

Figure Lengend Snippet: Antagonist compounds 1–3 , but not control 5 , inhibit NF-κB activation in ( A ) human TLR7-, ( B ) human TLR8- and ( C ) mouse TLR9-expressing HEK293 cells. Antagonist compounds do not inhibit NF-κB activation in ( D ) human TLR3- and ( E ) human TLR4-expressing HEK293 cells. HEK293 cells expressing human TLR3 and 4 and mouse TLR9 and human TLR7 and 8 XL-HEK293 cells were incubated with 10 µg/ml TLR3 agonist, 0.1 µg/ml TLR4 agonist, 5 µg/ml TLR9 agonist, 50 µg/ml TLR7 agonist and 50 µg/ml TLR8 agonist alone, respectively, and in combination with various concentrations of antagonist compounds 1–3 or control oligo 5 . After 18 h of incubation, supernatants were assessed for NF-κB activation using SEAP assays as described in ‘Materials and Methods’ section. The results are expressed as fold change in NF-κB activation compared with PBS control. Each value shown is mean of two wells ± SD. Data shown are representative of three independent experiments.

Article Snippet: Human embryonic kidney (HEK)293 cells stably expressing human TLR3, TLR4/CD14/MD-2 or mTLR9 and HEK293XL cells stably expressing human TLR7 or TLR8 were obtained from Invivogen (San Diego, CA, USA).

Techniques: Activation Assay, Expressing, Incubation

Journal: Nucleic Acids Research

Article Title: Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9

doi: 10.1093/nar/gkt078

Figure Lengend Snippet: Effect of antagonist compound 1 on ( A ) TLR7-, ( B ) TLR9- and ( C ) TLR4-mediated NF-κB activation in J774 murine macrophage cells. J774 cells were incubated with various concentrations of antagonist compound 1 for an hour, and then further incubated for 1 h after adding 50 µg/ml TLR7 agonist or 1 µg/ml TLR9 agonist. Antagonist compound 1 alone treatment of J774 cells was carried out at 20 µg/ml concentration. Nuclear extracts were then prepared and electrophoretic mobility shift assay was carried out as described in ‘Materials and Methods’ section. ( D ) Antagonist compound 1 inhibits TLR9 agonist-induced p38 MAPK activation in J774 cells. Cells were incubated with PBS, antagonist compound 1 (10 µg/ml), TLR9 agonist (1 µg/ml) or antagonist compound 1 plus TLR9 agonist. Cells were pre-incubated with antagonist compound 1 for 1 h followed by TLR9 agonist stimulation for 30 min. Whole cell lysates were prepared and analyzed by western blotting as described in ‘Materials and Methods’ section. Data shown are representative of two independent experiments.

Article Snippet: Human embryonic kidney (HEK)293 cells stably expressing human TLR3, TLR4/CD14/MD-2 or mTLR9 and HEK293XL cells stably expressing human TLR7 or TLR8 were obtained from Invivogen (San Diego, CA, USA).

Techniques: Activation Assay, Incubation, Concentration Assay, Electrophoretic Mobility Shift Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9

doi: 10.1093/nar/gkt078

Figure Lengend Snippet: Dose-dependent inhibition of ( A ) TLR7- and ( B ) TLR9-mediated induction of IL-12 and IL-6 by antagonist compounds 1 , 2 and 3 in mouse spleen cell cultures. Control oligo 5 has no effect on TLR7- and TLR9-mediated cytokine induction. ( C ) None of the antagonist compounds inhibited TLR4-mediated induction of IL-12 and IL-6. Spleen cells were incubated with TLR7 agonist (50 µg/ml), TLR9 agonist (1 µg/ml) or TLR4 agonist (1 µg/ml) in the absence or presence of various concentrations of antagonist compounds 1–3 or control oligo 5 for 24 h in triplicate wells. Cell culture supernatants were assessed for IL-12 and IL-6 levels by ELISA. Data shown are mean of triplicate wells ± SD and representative of three independent experiments.

Article Snippet: Human embryonic kidney (HEK)293 cells stably expressing human TLR3, TLR4/CD14/MD-2 or mTLR9 and HEK293XL cells stably expressing human TLR7 or TLR8 were obtained from Invivogen (San Diego, CA, USA).

Techniques: Inhibition, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nucleic Acids Research

Article Title: Design, synthesis and biological evaluation of novel antagonist compounds of Toll-like receptors 7, 8 and 9

doi: 10.1093/nar/gkt078

Figure Lengend Snippet: Antagonist compound 1 -treated NHP PBMCs show reduced cytokine production in response to ( A ) TLR7, ( B ) TLR8 and ( C ) TLR9, but not ( D ) TLR4, agonist stimulation. Cynomolgus monkeys ( N = 4) were injected s.c. with 1.5 mg/kg antagonist compound 1 and blood was collected at pre-administration and various post-administration time points. PMBCs were isolated and stimulated for 24 h with TLR7 (50 µg/ml), TLR8 (50 µg/ml), TLR9 (3 µg/ml) and TLR4 (0.1 µg/ml) agonists. Cell culture supernatants were analyzed for cytokine levels using a multiplex assay. Pre and Post indicate pre-administration and 48 h post-administration time points. Each individual data point indicates data for one animal, and black line indicates mean of all animals.

Article Snippet: Human embryonic kidney (HEK)293 cells stably expressing human TLR3, TLR4/CD14/MD-2 or mTLR9 and HEK293XL cells stably expressing human TLR7 or TLR8 were obtained from Invivogen (San Diego, CA, USA).

Techniques: Injection, Isolation, Cell Culture, Multiplex Assay