cli095  (Thermo Fisher)

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