Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Cytokine arrays of 143B-Luc cells, human macrophages, and co-culture CM. B IL-8 production during the co-culture of OS cells (143B-Luc, SJSA-1) and macrophages (THP-1 Mφ, HMDMs). C IL-8 expression in macrophages stimulated with OS-CM. D IL-8 expression in OS cells stimulated with macrophage CM. E UMAP plot of OS lung metastases. F UMAP plot of Iba1, CD163, and IL-8 expression in myeloid cell clusters. G Violin plot depicting IL-8 expression in Iba1 +/− or CD163 +/− myeloid cell clusters. CM conditioned medium, HMDMs human monocyte-derived macrophages, OS osteosarcoma, THP-1 Mφ THP-1-derived macrophage. Data are presented as mean ± standard deviation. **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Co-Culture Assay, Expressing, Derivative Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Cell viability assay for OS cells treated with or without recombinant IL-8 (rIL-8, 10 ng/mL) using Cell Counting Kit-8. B , C Scratch and invasion assays for OS cells treated with or without co-culture CM. The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. D Cell viability assay for OS cells treated with or without co-culture CM and with or without anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) using Cell Counting Kit-8. E , F Scratch and invasion assays for OS cells treated with or without co-culture CM and with or without IL-8 Abs (100 ng/mL). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM: conditioned medium, OS: osteosarcoma, ns: not significant. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Viability Assay, Recombinant, Cell Counting, Co-Culture Assay, Wound Healing Assay, Invasion Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Western blot assays for FAK phosphorylation in OS cells treated with co-culture CM and quantification of western blot bands. B Western blot assays for the phosphorylation of FAK (p FAK) in OS cells treated with co-culture CM + isotype IgG (IgG) or + anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) and quantification of western blot bands. The FAK phosphorylation levels were also compared between the same times. C Cell viability assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186, 1 µM) using Cell Counting Kit-8. D , E Scratch and invasion assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186) (1 µM). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM conditioned medium, FAK focal adhesion kinase, ns not significant, OS osteosarcoma. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Western Blot, Co-Culture Assay, Viability Assay, Cell Counting, Invasion Assay, Wound Healing Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Schematic showing the experimental design for subcutaneous transplantation of OS cells. 143B-Luc cells (2 × 10 6 /mouse) were subcutaneously transplanted in 6–7-week-old BALB/c nu/nu mice, and DMEM or co-culture CM was injected into the para-tumor thrice per week. B Tumor volume with or without co-culture CM measured thrice per week after tumor transplantation. C , D Tumor weight and image of the excised tumor with or without co-cultured CM 14 days after tumor transplantation. E , F Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with or without co-culture CM 14 days after tumor transplantation. Two fields of view per sample were randomly selected, and quantification was performed in 10 fields. E Ki-67-positive cells in the field were counted and indicated as Ki-67 labeling index. Scale bars represent 50 μm. G Schematic showing the experimental design for subcutaneous OS transplantation using anti-IL-8 antibodies (IL-8 Abs). H Tumor volume after pretreatment with co-culture CM with or without IL-8 Abs (10 µg/mouse) measured thrice per week after tumor transplantation. I and J Weight and image of the excised tumor pretreated with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. K , L Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. Scale bars represent 50 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, FAK focal adhesion kinase, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Transplantation Assay, Co-Culture Assay, Injection, Cell Culture, Immunohistochemistry, Labeling, Modification, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Schematic showing the experimental design for OS tail vein injection. 143B-Luc cells were treated with DMEM or co-culture CM for 12 h prior to tail vein injection; 143B-Luc cells (1 × 10 6 /mouse) were injected into the tail vein of 6–7 week-old BALB/c nu/nu mice. B IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with or without co-culture CM. C Hematoxylin and eosin staining of lung sections with or without co-culture CM and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. D Immunohistochemistry of Ki-67 positive cells in lung colonies with or without co-culture CM and quantification of the Ki-67 labeling index. Two colonies per sample were randomly selected, and quantification was performed for 10 colonies. Ki-67-positive cells in the colonies were counted and shown as the Ki-67 labeling index. Scale bars represent 100 µm. E Schematic showing the experimental design for OS tail vein injection using anti-IL-8 antibody. F IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with co-culture CM with or without IL-8 antibodies. G Hematoxylin and eosin staining of lung sections with co-culture CM with or without IL-8 Abs and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. H Immunohistochemistry of Ki-67-positive cells in lung colonies with co-culture CM with or without IL-8 Abs and quantification of Ki-67 labeling index. Scale bars represent 100 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, IVIS, in vivo imaging system, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Injection, Co-Culture Assay, Imaging, Staining, Immunohistochemistry, Labeling, Modification, In Vivo Imaging, Standard Deviation

Journal:

Article Title: Selective Inhibition of the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein by 1,25(OH) 2 Vitamin D 3

doi: 10.1016/j.molimm.2005.07.023

Figure Lengend Snippet: Effect of 1,25(OH)2D3 on chemotaxis to C5a, LTB4, formyl peptide and CXCL8. Purified DBP (100 nM) was treated for 5 min at 37°C with 10 nM 1,25(OH)2D3. Neutrophils (4 x 106/ml) were then preincubated for 40 min at 37°C with either DBP plus 1,25(OH)2D3 or buffer. Neutrophil movement then was assayed for 30 min at 37°C. Numbers represent mean ± SEM of 3 experiments using cells from different donors.

Article Snippet: Recombinant CXCL8 (IL-8) was obtained from R&D Systems (Minneapolis, MN).

Techniques: Chemotaxis Assay, Purification