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: bioRxiv

Article Title: Interrogating the genus Yersinia to define the rules of lipopolysaccharide lipid A structure associated with pathogenicity

doi: 10.1101/2025.03.03.641127

Figure Lengend Snippet: (A) Initial snapshot of the (TLR4/MD-2) 2 complex bound to m/z 1768 (left). Final snapshots of each one side of the (TLR4/MD-2) 2 complex bound to m/z 1768 (right). Labelled basic amino acid residues observed to make significant hydrogen-bonding interactions with m/z 1768 are shown in licorice format, coloured in blue. (B) Root mean square deviation (RMSD) of TLR4 dimer backbone atoms. (C) Buried solvent accessible surface area (SASA) between MD-2-lipid A species and TLR4. (D) Contacts between lipid A species and (TLR4/MD-2) 2 . (E) Overlaid every 200 ns simulation snapshots of F126 side chain and its orientation with respect to the MD-2:lipid A species. The pseudo trajectory was made of two aligned MD-2:lipid monomer frames for each system. The F126 side chain is shown in licorice representation and coloured according to simulation time (red-white-blue for 0 to 2,000 ns). (F) RMSD of MD-2 backbone loop region after alignment on the entire MD-2 backbone. (G) Porcupine plot of TLR4 dimer alpha carbons corresponding to the most dominant motion (PCA1). The colours corresponds to distances as labelled inset. In panel (A) and (E) protein is shown in cartoon representation: TLR monomers in violet and red, MD-2 dimers shown in grey. Lipid A species and side chains are shown in licorice representation (cyan – carbon, red – oxygen, blue – nitrogen, brown – phosphorus). All values in panels (B)-(D) and (F) were averaged over last 200 ns and across both dimers.

Article Snippet: To gain insights into the potential of the different lipid A molecular species as TLR4/MD-2 agonists, we used explicitly solvated atomic-resolution molecular dynamics (MD) simulations to assess the interaction between the lipid A molecular species and the TLR4 2 /MD-2 2 complex, and with the F126 residues within the MD-2 loop encompassing 120 to 129 residues.

Techniques: Solvent

Journal: bioRxiv

Article Title: Interrogating the genus Yersinia to define the rules of lipopolysaccharide lipid A structure associated with pathogenicity

doi: 10.1101/2025.03.03.641127

Figure Lengend Snippet: (A) Surface expression of TLR4 as measured by mean fluorescence intensity (MFI) on iBMDMs non-infected (ni) or exposed to live E. coli , pathogenic yersiniae, YeO8 and YeO3, and non- pathogenic yersiniae , 1A strain 0902, Y. aldovae, Y. nurmii , grown at 21°C (denoted as 21) and 37°C (denoted as 37). (B)) Surface expression of TLR4 as measured by MFI on iBMDMs non-infected (ni) or exposed to live YeO8, chimeric YeO8 strain expressing a non-pathogenic lipid A, strain YeO8NPL, and YeO8 lpxR mutant (Δ lpxR , strain YeO8-Δ lpxR ) grown at 21°C (denoted as 21) and 37°C (denoted as 37). (C) TNFα secretion by infected iBMDMs with YeO8,and YeO8NPL.“c” denotes bacteria without the virulence plasmid. Strains were grown at 21°C (denoted as 21) and 37°C (denoted as 37). (D) TNFα secretion by iBMDMs challenge with 10 ng/ml of repurified LPS from YeO8, YeO8NPL, and YeO8 lpxR mutant (Δ lpxR , strain YeO8-Δ lpxR ) grown at 37°C. Data are presented as mean ± SD (n□=□3). In panels A and B, **** P □≤□0.0001; ns, P □>□0.05 for the comparisons against non-infected cells, and # P □≤□0.0001 for the comparisons against YeO8 grown at 37°C using One□way ANOVA with Bonferroni contrast for multiple comparisons test. In panels C and D, **** P □≤□0.0001; ns, P □>□0.05 for the indicated comparisons using One□way ANOVA Dunnett’s multiple comparisons test.

Article Snippet: To gain insights into the potential of the different lipid A molecular species as TLR4/MD-2 agonists, we used explicitly solvated atomic-resolution molecular dynamics (MD) simulations to assess the interaction between the lipid A molecular species and the TLR4 2 /MD-2 2 complex, and with the F126 residues within the MD-2 loop encompassing 120 to 129 residues.

Techniques: Expressing, Fluorescence, Infection, Mutagenesis, Bacteria, Plasmid Preparation

Journal: Biomolecules

Article Title: Novel Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling

doi: 10.3390/biom14111441

Figure Lengend Snippet: Comparison of protein expression in Vehicle- and 3α,5α-THP-treated P rats. 3α,5α-THP does not alter the expression of key TLR4-associated proteins. To further clarify the effects of 3α,5α-THP on TLR4 signaling, various proteins involved in the TLR4 pathway were tested. Male and female P rats were treated with 3α,5α-THP (15 mg/kg; 30 min) or vehicle (45% w / v 2-hydroxypropyl-β-cyclodextrin; 30 min), as previously described. There were no significant sex or treatment effects in protein expression in the hippocampi of P rats of the listed proteins.

Article Snippet: AutoDock Vina (Version 1.2.0) was then used for the docking TLR4:MD-2 and the calculation of docking scores [ ].

Techniques: Comparison, Expressing

Journal: Biomolecules

Article Title: Novel Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling

doi: 10.3390/biom14111441

Figure Lengend Snippet: 3α,5α-THP has no effect on TLR4 binding to MD-2 in the hippocampus of male and female P rats. ( A , B ) Hippocampus whole lysate was immunoblotted for the presence of MD-2 in vehicle- (45% w / v 2-hydroxypropyl-β-cyclodextrin; 30 min) and 3α,5α-THP-treated (15 mg/kg; 30 min) P rats and densiometric comparison of the effect of 3α,5α-THP on MD-2 expression. ( C , E ) Immunoprecipitation of TLR4/MD-2 and densiometric comparison of MD2 between vehicle and 3α,5α-THP-treated P rats (male: t -test, p = 0.56, n = 4/group female: ( t -test, p = 0.46, n = 4/group) ( D , F ). Western blot original images are in the . ns: no significant difference.

Article Snippet: AutoDock Vina (Version 1.2.0) was then used for the docking TLR4:MD-2 and the calculation of docking scores [ ].

Techniques: Binding Assay, Comparison, Expressing, Immunoprecipitation, Western Blot

Journal: Biomolecules

Article Title: Novel Inhibitory Actions of Neuroactive Steroid [3α,5α]-3-Hydroxypregnan-20-One on Toll-like Receptor 4-Dependent Neuroimmune Signaling

doi: 10.3390/biom14111441

Figure Lengend Snippet: 3α,5α-THP docks TLR4-bound MD-2 and inhibits Lipid A binding to MD-2 in SPR studies. ( A ) Molecular docking shows that 3α,5α-THP favors binding to MD-2, with multiple docking poses of 3α,5α-THP found within the MD-2 binding pocket. ( B ) The top pose of 3α,5α-THP in MD-2 shows multiple π-alkyl bonds formed with key MD-2 residues. ( C ) Two-dimensional representation of 3α,5α-THP binding MD-2, depicting the amino acid interactions. ( D ) SPR studies show Lipid A binds immobilized MD-2 with a K D = 4.3 ± 0.5 µM (k a = 1.5 × 10 2 M −1 S −1 , k d = 6.78 × 10 −4 S −1 ). ( E ) SPR competition assay showing that 3α,5α-THP competitively binds MD2, as increasing concentrations of 3α,5α-THP decrease Lipid A binding ( D ).

Article Snippet: AutoDock Vina (Version 1.2.0) was then used for the docking TLR4:MD-2 and the calculation of docking scores [ ].

Techniques: Binding Assay, Competitive Binding Assay

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