tlr2 antagonist c29  (Chemdiv Inc)

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 TLR2 signaling, ligand priority and contact point of the two antagonists CU-CPT22 and C29. b HEK293 TLR2/1 or 2/6 reporter cells were stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 or Pam CSK 4 . Dashed lines represent the six time points that were used to generate concentration-effect curves ( c ). Baseline-corrected DMR recordings are mean + SEM and representative of three biologically independent experiments. c Sigmoidal concentration-effect curves resulting from DMR traces of n biologically independent experiments ( c : n = 3 biological replicates except Pam 3 CSK 4 log 3, log 2, log 1 n = 5, Pam CSK 4 log 0, 50 min n = 2) (Mean ± SEM). Concentration-effect curves of DMR data were generated by the response at six different time points ( b ). d HEK293 TLR2 reporter cells were stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 or Pam CSK 4 . HEK293 TLR2/1 or 2/6 reporter cells were preincubated with 50 µM of the TLR2 antagonist ( e ) CU-CPT22 or ( f ) C29 stimulated with the indicated concentrations (ng/ml) of Pam 3 CSK 4 or Pam CSK 4 . Calculated pharmacological parameters of the concentration-effect curves ( c ) are depicted in Table . 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/q83w265.

Article Snippet: The TLR2 antagonist C29 was obtained from ChemDiv (San Diego, USA).

Techniques: Concentration Assay, 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 parameters of Pam 3 CSK 4 - and Pam 2 CSK 4 -induced DMR at six selected time points in HEK293 TLR2/1 and TLR2/6 reporter cells

Article Snippet: The TLR2 antagonist C29 was obtained from ChemDiv (San Diego, USA).

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 HaCaT cells were stimulated with LPS E. coli (1000 ng/ml), Pam 3 CSK 4 (1000 ng/ml) or Pam 2 CSK 4 (1000 ng/ml). b HEK293 TLR2/1 reporter cells were stimulated with Pam 3 CSK 4 (100 ng/ml) or Pam 2 CSK 4 (100 ng/ml). c HaCaT cells were preincubated with 50 µM of the TLR2 antagonists CU-CPT22 or C29 stimulated with Pam 3 CSK 4 (1000 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.

Article Snippet: The TLR2 antagonist C29 was obtained from ChemDiv (San Diego, USA).

Techniques:

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: Screening of a receptor target for SAA1 in HSCs (A and B) LX-2 and PRHSCs were stimulated with rhSAA1 or rmSAA1, respectively, for 24 hr, and mRNA levels of TLR2, TLR4, FPR2, RAGE, and SR-B1 were determined by RT-qPCR. (C and D) Representative western blot analysis of receptors TLR2, TLR4, FPR2, RAGE, and SR-B1 after rhSAA1 and rmSAA1 treatment of LX-2 and PRHSCs, respectively. (E and F) LX-2 and PRHSCs were transfected with reporter plasmid for NF-κB. Twenty four hr later, the medium was changed and cells were pretreated with inhibitors of TLR2 (CU-CPT22, 1 μM), RAGE (FPS-ZM1, 0.5 μM), TLR4 (TAK 242, 5 nM), and FPR2 (WRW4, 0.25 μM) and then stimulated with rhSAA1 and rmSAA1, respectively, diluted in serum-free DMEM. Twelve hr later, luciferase activity was measured in fold induction after normalization with Renilla luciferase (Rluc) control. (G and H) Co-immunoprecipitation (co-IP) experiment showing the interaction between SAA1 and TLR2 determined by silver staining and western blot analysis. (For detailed information please see section). (n = 3). Where applicable, data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and (n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Silver Staining

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: TLR2 serves as a chemotactic receptor for SAA1 and mediates migration of HSCs (A) Schematic of the TLR2 gene knockout strategy. The Cas9/sgRNA(s) target site(s) are indicated in red and were confirmed by sequencing. (B) Sequencing result of targeted region. (C) Confirmation of TLR2 gene KO at protein level determined by western blot. (D and E) WT and TLR2 −/− LX-2 cells (D) and WT and siPRHSCs (E) were transfected with reporter plasmid for NF-κB. Luciferase activity was measured as fold induction in comparison to untreated control (See also section for detail information). (F and G) Representative images showing agarose spot and Transwell migration assays of SAA1-treated WT LX-2, TLR2 −/− LX-2 cells, and non-treated control. (H and I) Representative images for immunostaining of TLR2 (H) and α-SMA + cells (I) in CU-CPT22- or VL-treated samples as shown in CCl 4 and CI injury models. Bar graph represents quantification of IHC images per 5 field(s). (J and K) Immunofluorescence images showing co-localization of SAA1 and HSCs in CU-CPT22-treated and control (VL) samples in CCl 4 (J) and CI injury models (K). (Scale bar represents 100 μm for CCl 4 injury, 200 μm for CI injury, and 50 μm for inset). Data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.0001 (n = 3). (For detailed TLR2 knockout strategy, see section).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Migration, Gene Knockout, Sequencing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Comparison, Control, Immunostaining, Immunofluorescence, Knock-Out

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis guides homing of transplanted LX-2 toward injury site(s) (A) Schematic representation of experimental design. (B) Bioluminescence imaging of transplanted cells entrapped to the liver 24 hr after transplantation in CCl 4 injury model, (A) represents sham operated mice, (B) represents SAA1siRNA2-treated mice transplanted with WT LX-2, (C) represents Neg-siRNA-treated mice transplanted with TLR2 −/− LX-2, and (D) represents control mice transplanted with WT LX-2 cells. (C and D) Representative confocal immunofluorescence images showing the recruitment of transplanted WT and TLR2 −/− LX-2 cells at injury locus after CCl 4 and CI injuries induced in SAA1-siRNA2, Neg-siRNA, and control samples, respectively. Bar graph represents quantification of relative number of homed cells at injury site(s). (Scale bar represents 100 μM). Data represent mean ± SEM ∗p < 0.01, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001(n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Imaging, Transplantation Assay, Control, Immunofluorescence

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis induces Rac GTPase-mediated actin reorganization and migration of HSCs (A) Time course of GTPase-Rac1 activation(s) in LX-2 cells at indicated time interval (B and C) Basal and stimulated levels of GTPase-Rac1 in WT and TLR2 −/− cells as determined by pull-down assays (D and E) WT and TLR2 −/− LX-2 cells were pretreated with NSC 23766 (50 μΜ), and activation of GTP-Rac1 was determined by pull-down assay (D), and MLCK and p-MLC at Ser19 activity was determined by western blotting (E). (F and G) Migration assays showing pretreatment of the cells with NSC 23766 (50 μΜ) attenuated their migration(s) in agarose spot (F) and Transwell (G) assays. (H) Time course of PI3K activations after treatment of LX-2 cells at indicated time points. (I and J) WT and TLR2 −/− cells were pretreated with LY294002 (10 μΜ), and the phosphorylation of PI3K at p85 subunits was determined by western blot analysis, and the activations of Rac GTPase were determined by pull-down assay. (K and L) Migration assays showing pretreatment of the cells with LY294002 attenuated their migration(s) in agarose spot (K) and Transwell (L) assays. Photographs are representatives of (n = 6). (Scale bar represents 200 μm). Data represent mean ± SEM ∗p < 0.01 and ∗∗p < 0.001 (n = 3). Photographs are representatives of (n = 6). (Scale bar represents 200 μm). Data represent mean ± SEM ∗p < 0.01 and ∗∗p < 0.001 (n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Migration, Activation Assay, Pull Down Assay, Activity Assay, Western Blot, Phospho-proteomics

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis mediates increased deposition of ECM at injury sites and induces chemokines secretion in activated HSCs (A and B) Sirius red staining of ECM deposition at injury locus in CCl 4 and Cl-induced models. (C) ELISA detection of MCP-1, IL-8, and RANTES secretion from LX-2 cells after treatment of the cells with rhSAA1 for 24 hr. (D) mRNA levels of MCP-1, IL-8, and RANTES. (E) Protein-level detection of MCP-1, IL-8, and RANTES. (Scale bar represents 100 μm). Data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01 (n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet:

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Marker, Polymer, Plasmid Preparation, Recombinant, Protease Inhibitor, Saline, Enzyme-linked Immunosorbent Assay, Activation Assay, Silver Staining, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Transfection, Gene Expression, Reporter Assay, Staining, Over Expression, Software, Imaging, Live Cell Imaging, Microscopy