cli095  (InvivoGen)

Journal: Frontiers in Immunology

Article Title: Carbohydrate fatty acid monosulphate ester adjuvant enhances the immunogenicity of influenza antigens via TLR4/2-dependent mechanisms

doi: 10.3389/fimmu.2026.1787181

Figure Lengend Snippet: CMS activates DCs through TLR4 and TLR2-dependent mechanisms. (A) Heat map representation depicting the expression of different TLRs comparing unstimulated DCs vs CMS-stimulated DCs. (B) THP-1 human monocytic cell line stably expressing a secreted embryonic alkaline phosphatase (SEAP) reporter inducible by NF-kB was treated for 24 hrs with CMS at the indicated concentrations. The activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI−Blue™ Solution. Results of THP1 MD2-CD14-TLR4, THP1 and THP1 KO-TLR4 are shown (mean ± SD, n=4). (C, E) Effect of CLI095 (TLR4 pathway inhibitor), or TL2-C29 (TLR2 pathway inhibitor), compared to DMSO (solvent control) on the expression of CMS-induced DC maturation markers. Scatter plots representing the mean ± SD (n = 8 donors) values of CD80, CD86, and HLA-DR are presented. (D, F) Inhibitory effect of CLI095 or TL2-C29 on the secretion (mean ± SD, n = 8) of IL-8, IL-6, and IL-12p70 (all in pg/ml) by CMS-stimulated DCs. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 as analyzed by one-way ANOVA with Tukey’s multiple comparisons post-test (C-F) . CA, cells alone; DMSO, dimethyl sulfoxide; SD, standard deviation.

Article Snippet: For TLR signaling inhibition, DCs were pre-incubated with CLI095 (3 μM), TL2-C29 (200 μM), CU-CPT9a (10 μM) (all from Invivogen) or 0.1% dimethyl sulfoxide prior to CMS stimulation (125 μg/ml).

Techniques: Expressing, Stable Transfection, Activation Assay, Activity Assay, Solvent, Control, Standard Deviation, FACS

Journal: Acta biochimica et biophysica Sinica

Article Title: TLR4 mediates lipotoxic β-cell dysfunction by inhibiting the TMEM24/PI3K/AKT pathway.

doi: 10.3724/abbs.2025045

Figure Lengend Snippet: Figure 2. Lipotoxicity mediated by TLR4 is closely linked to TMEM24 (A) β-Cells were treated with a TLR4 inhibitor (CLI095) or agonist (LPS), and TLR4 protein levels were detected by western blot analysis. (B) β-Cells were treated with CLI095 or LPS, and TMEM24 protein levels were detected by western blot analysis. (C) Insulin secretion from β-cells treated with CLI095 or LPS. (D) Apoptosis rates were determined by flow cytometry across groups. (E) β-Cells were transfected with siRNA for 24 h. TLR4 knockdown efficiency was then determined by western blot analysis. (F) TMEM24 protein expression across groups was detected by western blot analysis. (G) Insulin secretion across groups. (H) Apoptosis rates were determined by flow cytometry across groups. *P < 0.05 vs the NC group and #P < 0.05 vs the PA group. Data are presented as the mean ± SD (n = 3).

Article Snippet: Then, the cell culture medium was removed, and 1.0 mM PA (Sigma-Aldrich), 1.0 μM CLI095 (MCE), 1.0 μg/mL LPS (Sigma-Aldrich), or 20 μm YS-49 (activator of the PI3K/AKT pathway; HY-15477; MCE) was added and incubated for 24 h. TUNEL assay Apoptosis was assessed by flow cytometry as previously described [30].

Techniques: Western Blot, Flow Cytometry, Transfection, Knockdown, Expressing

Journal: Theranostics

Article Title: IL4 receptor targeting enables nab-paclitaxel to enhance reprogramming of M2-type macrophages into M1-like phenotype via ROS-HMGB1-TLR4 axis and inhibition of tumor growth and metastasis

doi: 10.7150/thno.92672

Figure Lengend Snippet: Internalization and cytotoxicity of IL4R-Abx in M2 macrophages. (A) Immunofluorescence analysis of the internalization. M2 macrophages were incubated using 2 μg/mL of fluorescein isothiocyanate (FITC) dye (green)-labeled Abx, Ctrl-Abx, and IL4R-Abx for 2 h with or without pre-treatment with 5-(n-ethyl-n-isopropyl) amiloride (EIPA) (50 μM), chlorpromazine (CPZ) (50 μM), anti-IL4R antibody (100 μM), and IgG (100 μM) for 30 min. Nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI) (blue), and merging of images was performed. Scale bars, 20 μm. (B) Flow cytometry analysis of internalization. M2 macrophages were treated as described in (A), and the mean fluorescence intensity (MFI) was quantified using a flow cytometer. Data are expressed as mean ± standard deviation (SD) in the three separate experiments. P values of Ctrl-Abx and IL4R-Abx compared to Abx in each group are shown on the top of bars. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant through one-way analysis of variance (ANOVA) followed by Tukey's multiple post hoc test. (C) Cytotoxicity assay. M2 macrophages were treated with 5 μg/mL of Abx, Ctrl-Abx, and IL4R-Abx for 24 h with or without pre-treatment with 500 nM of CLI095 for 30 min and subjected to cytotoxicity assays. Data are expressed as mean ± SD in the three separate experiments. P values compared to untreated control in each group are shown on the top of bars. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant by two-way ANOVA followed by Bonferroni multiple post hoc test.

Article Snippet: To inhibit or block HMGB1 and TLR4 activity, cells were incubated for 30 min with 10 μg/mL of anti-HMGB1 antibody (Arigo Biolaboratories, Hsinchu, Taiwan) and 500 nM of CLI095 (Thermo Fisher), respectively, before treatments with a conditioned medium (CM) of IL4R-Abx- and Abx-treated M2 macrophages.

Techniques: Immunofluorescence, Incubation, Labeling, Staining, Flow Cytometry, Fluorescence, Standard Deviation, Cytotoxicity Assay, Control

Journal: Theranostics

Article Title: IL4 receptor targeting enables nab-paclitaxel to enhance reprogramming of M2-type macrophages into M1-like phenotype via ROS-HMGB1-TLR4 axis and inhibition of tumor growth and metastasis

doi: 10.7150/thno.92672

Figure Lengend Snippet: IL4R-Abx reprograms M2 macrophages into M1-like phenotype via the ROS-HMGB1-TLR4 axis. (A) M2 macrophages were incubated with 5 μg/mL of Abx and IL4R-Abx for 12 and 24 h. Concentrations of HMGB1 released into the conditioned medium were determined by ELISA. (B) M2 macrophages were incubated with 5 μg/mL of Abx and IL4R-Abx for 24 h with or without pre-treatment with 50 mM of NAC for 30 min. The conditioned medium was collected, concentrated, and subjected to western blotting using an anti-HMGB1 antibody. HMGB1 protein band intensity was quantified using Image J software. (C) M2 macrophages were incubated with Abx and IL4R-Abx for 24 h with or without pre-treatment with NAC. Cells were incubated with anti-HMGB1 antibody (red). Nucleus was stained with DAPI (blue), and images were merged. Boxes represent the enlarged area. Scale bars, 40 µm. (D-I) M2 macrophages were incubated using Abx or IL4R-Abx for 12 h with or without pre-treatment of anti-HMGB1 antibody (10 μg/mL, CM+αHMGB1) and CLI095 (500 nM, CM+CLI095) for 30 min. The CM of M2 macrophages was collected and treated to other sets of M2 macrophages for 24 h. Relative mRNA levels of M1-macrophage markers, such as il12p40 (D) , il-6 (E) , and ifn-γ (F) , and M2-macrophage markers, such as fizz-1 (G) , tgf-β (H) , and il-10 (I) , were measured via qRT-PCR analysis. Data are expressed as mean ± SD in the three separate experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant by one-way ANOVA followed by Tukey's multiple post hoc test. (-), untreated M2-macrophage control (black bar).

Article Snippet: To inhibit or block HMGB1 and TLR4 activity, cells were incubated for 30 min with 10 μg/mL of anti-HMGB1 antibody (Arigo Biolaboratories, Hsinchu, Taiwan) and 500 nM of CLI095 (Thermo Fisher), respectively, before treatments with a conditioned medium (CM) of IL4R-Abx- and Abx-treated M2 macrophages.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Software, Staining, Quantitative RT-PCR, Control