Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 5. sRAGE and anti-RAGE inhibit CXCL8- and TNF-- but not ATRA-induced type X collagen expression. Primary normal human knee articular chondrocytes (donor ages, 29–62) (5 105 cells/six-well dish) were stimulated with CXCL8 (A), ATRA (B), or TNF- (C) in the presence or absence of 1 g/ml sRAGE or 20 g/ml anti-RAGE. SDS- PAGE and Western blotting analysis for type X collagen were performed on cell lysates at 5 days in culture. These data are representative of results from six normal human donors.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Expressing, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 3. RAGE expression and inducible S100A11 secretion in cul- tured human articular chondrocytes. A, RAGE expression. First-passage human knee articular chondrocytes (5 105 cells/six-well dish) from six normal donors (age range, 29–62), were prepared as described in Mate- rials and Methods, were studied for RAGE expression by flow cytometric analysis. The panels, taken from studies of one normal donor representative of all the donors, depict binding of isotype control IgG (open histogram) and additionally of anti-RAGE IgG without stimulation or in response to CXCL8, TNF-, and ATRA for 24 h (solid histograms). In normal chon- drocytes, constitutive RAGE expression was detected in 59 9% of cells, and there were no significant changes in the proportions of RAGE-express- ing chondrocytes in response to CXCL8, TNF-, or ATRA, as seen in the representative results from the single donor demonstrated here. B, S100A11 expression and multimerization in chondrocytes. SDS-PAGE/ Western blotting analysis was performed on 1 g of human recombinant S100A11 isolated from transfected 293 cells as described in Materials and Methods (left side of figure), and on 30-g aliquots of protein precipitated from conditioned medium of first-passage normal human knee chondro- cytes (right side of figure) (donor ages, 29–62). The chondrocytes were prepared under the same conditions as for A above, and stimulated for 24 h by addition of S100A11, CXCL8, TNF-, or ATRA, at which time con- ditioned media were studied. Results are representative of those from six different normal human donors, as in A.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Expressing, Binding Assay, Control, SDS Page, Western Blot, Recombinant, Isolation, Transfection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products.

doi: 10.4049/jimmunol.175.12.8296

Figure Lengend Snippet: FIGURE 7. Schematic of RAGE signaling in the induction of chondro- cyte hypertrophy. This schematic depicts the induction by CXCL8 and TNF- of secretion of S100A11 (and likely other chondrocyte-expressed RAGE ligands not limited to calgranulins). The paradigm further depicts subsequent RAGE signaling critically transduced by p38 MAPK pathway activation as a central event in chondrocyte hypertrophy associated with low-grade inflammation in the OA joint articular cartilage.

Article Snippet: All-trans retinoic acid (ATRA), and human recombinant CXCL8 and TNF- were from R&D Systems, and rabbit polyclonal Abs to type X collagen were from Calbiochem. mAb to RAGE was from Chemicon International.

Techniques: Activation Assay

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: Onco

Article Title: Angiodrastic Chemokines Production by Colonic Cancer Cell Lines

doi: 10.3390/onco2020006

Figure Lengend Snippet: Figure 3. Comparison of CXCL8 secretion over time by the two cell lines after incubation with cancer or normal serum. Mean and standard error (vertical bars) of five separate experiments are presented.

Article Snippet: Commercially available recombinant human CXCL8, CXCL4, CXCL6, and VEGF were obtained (R&D Systems Inc., Mineapolis, MN, USA) and used for generating standard curves.

Techniques: Comparison, Incubation