Journal: Scientific Reports

Article Title: CXCL8 mediates macrophage migration in canine oral malignant melanoma

doi: 10.1038/s41598-025-22749-x

Figure Lengend Snippet: mRNA expression levels of chemokine in seven canine melanoma cell lines. a CCL5 , b CX3CL1 , c CSF-1 , d IL-34 , ( e ) CXCL8 , and ( f ) CCL2 . The relative expression level was normalized to the RPL32 gene . g Correlation between the number of migratory RAW264.7 and CXCL8 mRNA expression levels. h Production of CXCL8 in seven canine melanoma cell lines. i Correlation between the number of migratory RAW264.7 and CXCL8 secretion. All data are presented as means ± SD of three independent experiments.

Article Snippet: The recombinant canine CXCL8 protein (NBP2-34,908, Novus Biologicals, Centennial, CO, USA) was supplemented at a final concentration of 200 ng/mL into the CM.

Techniques: Expressing

Journal: Scientific Reports

Article Title: CXCL8 mediates macrophage migration in canine oral malignant melanoma

doi: 10.1038/s41598-025-22749-x

Figure Lengend Snippet: a , b Relative number of RAW264.7 cells migrated by CMeC1 wild-type CM or CMeC1 CXCL8 knockout CM. c Relative number of RAW264.7 cells migrated by CMeC1 CM. The CM of CMeC1 wild-type cells were treated with anti-dog CXCL8 (1 μg/ml) or mouse IgG isotype (1 μg/ml). All data are presented as means ± SD of three independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: The recombinant canine CXCL8 protein (NBP2-34,908, Novus Biologicals, Centennial, CO, USA) was supplemented at a final concentration of 200 ng/mL into the CM.

Techniques: Knock-Out

Journal: Scientific Reports

Article Title: CXCL8 mediates macrophage migration in canine oral malignant melanoma

doi: 10.1038/s41598-025-22749-x

Figure Lengend Snippet: Relative number of RAW264.7 cells migrated by CMeC1 CM. The CM of CMeC1 wild-type or CXCL8 knockout cells were treated with dog CXCL8 recombinant protein (200 ng/ml). All data are presented as means ± SD of three independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: The recombinant canine CXCL8 protein (NBP2-34,908, Novus Biologicals, Centennial, CO, USA) was supplemented at a final concentration of 200 ng/mL into the CM.

Techniques: Knock-Out, Recombinant

Journal: Scientific Reports

Article Title: CXCL8 mediates macrophage migration in canine oral malignant melanoma

doi: 10.1038/s41598-025-22749-x

Figure Lengend Snippet: Relative number of canine macrophage DH82 cells migrated by CMeC1 CM. The CM of CMeC1 wild-type or CXCL8 knockout were treated with anti-dog CXCL8 (1 μg/ml) or mouse IgG isotype (1 μg/ml). All data are presented as means ± SD of four independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: The recombinant canine CXCL8 protein (NBP2-34,908, Novus Biologicals, Centennial, CO, USA) was supplemented at a final concentration of 200 ng/mL into the CM.

Techniques: Knock-Out

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Functional in vitro activity of dual B7-H3-CXCR2 CAR T cells compared to single CAR B7-H3 CAR T cells . The ability of a chemokine receptor (CXCR2) to improve the activity of canine B7-H3 CAR T cells when the two molecules were co-expressed by transduced T cells was examined using in vitro studies. In A , the diagram of the dual B7-H3-CXCR2 construct is depicted. In B , the transduction efficiency of canine T cells with the dual CAR construct was evaluated by measuring L-protein binding (scFv detection) and with anti-huCXCR2 immunostaining, demonstrating expression of both molecules, along with the distribution of CD4 and CD8 CAR T cells. Flow cytometry dot plots showing percentage positive for each population. In C , to assess tumor target expression of a key chemokine (CXCL8) ligand for CXCR2, secretion of CXCL8 by 7 different canine OS tumor cell lines was measured, demonstrating that 6 of the 7 lines secreted large amounts of CXCL8 spontaneously. The ability of the huCXCR2 molecule to bind and become activated by canine CXCL8 was confirmed in D , using a transwell migration assay as described in Methods, with sustained migration of B7-H3-CXCR2 CAR T cells to a canine CXCL8 gradient. Serum containing medium was used as a positive control. In E , cytokine secretion following target Abrams cell engagement for 24 h was assessed, and revealed significantly greater production of both IFN-g and IL-2 with B7-H3-CXCR2 CAR T cells compared to B7-H3 CAR T cells, at equivalent E:T ratios (20:1)

Article Snippet: Migration was assessed using 8 μm transwell plates (Corning), with the lower chamber containing 25 ng/ml recombinant canine CXCL8 (R&D Systems) in serum free DMEM.

Techniques: Functional Assay, In Vitro, Activity Assay, Construct, Transduction, Protein Binding, Immunostaining, Expressing, Flow Cytometry, Transwell Migration Assay, Migration, Positive Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Transcriptomic comparison of B7-H3 and B7-H3-CXCR2 CAR T cells . RNA sequencing was performed to help elucidate in greater detail the functional differences between the two CAR T cells. In A , principal component analysis (PCA) plots depict B7-H3 CAR T cells, B7-H3-CXCR2 CAR T cells, and non-transduced T cells from 3 different dogs with biological replicates shown connected by lines. In B , volcano plots depict significantly upregulated (red) and downregulated (blue) genes in B7-H3-CXCR2 vs B7-H3 CAR T cells. Gene set enrichment analysis (GSEA) in C revealed significant upregulation (right of central axis) of pathways related to RNA processing and myc targets, along with downregulation of certain G-protein coupled receptor activities. Constitutive secretion of CXCL8 by B7-H3-CXCR2 CAR T cells in the absence of target cells was observed in panel D

Article Snippet: Migration was assessed using 8 μm transwell plates (Corning), with the lower chamber containing 25 ng/ml recombinant canine CXCL8 (R&D Systems) in serum free DMEM.

Techniques: Comparison, RNA Sequencing, Functional Assay

Journal: Respiratory Research

Article Title: Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

doi: 10.1186/1465-9921-14-88

Figure Lengend Snippet: CXCL8 protein is enhanced by HRV+CSE compared to CSE or HRV alone in HBE cells. HBE cells from 6 HBE cell donors to be used for siRNA experiments were treated with medium control, CSE, HRV or HRV+CSE for 24 h and cell supernatants were assessed for CXCL8 protein release. Data are presented as mean ± SEM (n=6). Asterisks denote a significant difference between the specified treatments (*** p<0.001).

Article Snippet: The following reagents were purchased from the indicated suppliers: bronchial epithelial cell basal medium and additives to create serum-free bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD); WI-38 cells and HRV type 16 (American Type Culture Collection, Manassas, VA); 3R4F research grade cigarettes (College of Agriculture Reference Cigarette Program, University of Kentucky) firefly luciferase reporter plasmid pGL4.10[ luc 2] and passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium Inc., Hayward, CA); TransIT-LT1 transfection reagent (Mirus, Madison, WI); anti-AUF-1 antibody (#07-260, Upstate curtsey of EMD Millipore, Billerica, MA); anti-KHSRP antibody (#Ab83291, Abcam, Toronto, ON, Canada); anti-HuR antibody (#A-21277, Molecular Probes courtesy of Life Technologies, Burlington, ON, Canada); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (AbD Serotec, Raleigh, NC); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA); HRP-conjugated anti-rabbit antibody (GE Healthcare Biosciences, Piscataway, NJ); enhanced chemiluminescent (ECL) substrate reagent (GE Healthcare Biosciences, Piscataway, NJ); Lowry DC protein assay (Biorad Laboratories, Mississauga, ON); recombinant CXCL8 protein (R & D Systems, Minneapolis, MN); siRNA targeting AUF-1 (S100300454 and S102653665), KHSRP (S100300587 and S100054691) and HuR (S100300139 and S103246551) (Qiagen, Toronto, ON, Canada); medium GC negative control non-targeting siRNA, OptiMEM reduced serum media and Lipofectamine RNAiMAX transfection reagent (Invitrogen, Burlington, ON, Canada).

Techniques: Control

Journal: Respiratory Research

Article Title: Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

doi: 10.1186/1465-9921-14-88

Figure Lengend Snippet: Effects of AUF-1-targeting siRNA on CXCL8 protein expression in HBE cells. HBE cells were treated for 24 h prior to harvesting cell supernatants for analysis with ELISA and whole cell lysates for analysis with immunoblotting. To determine the level of AUF-1 protein knock-down membranes were probed with a specific AUF-1 antibody recognizing 4 isoforms of AUF-1, then were subsequently stripped and re-probed with an antibody to GAPDH to ensure equal protein loading (A ; representative of 3 separate experiments ) . Densitometry analysis with % knock-down relative to HRV+CSE alone is also shown for each isoform of AUF-1 (A ; n=3 ) . Supernatants were analyzed for CXCL8 protein (B ; n=3 ) . Data are presented as mean ± SEM. Lipid denotes transfection reagent alone. NT denotes non-targeting control siRNA. ns = not significant.

Article Snippet: The following reagents were purchased from the indicated suppliers: bronchial epithelial cell basal medium and additives to create serum-free bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD); WI-38 cells and HRV type 16 (American Type Culture Collection, Manassas, VA); 3R4F research grade cigarettes (College of Agriculture Reference Cigarette Program, University of Kentucky) firefly luciferase reporter plasmid pGL4.10[ luc 2] and passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium Inc., Hayward, CA); TransIT-LT1 transfection reagent (Mirus, Madison, WI); anti-AUF-1 antibody (#07-260, Upstate curtsey of EMD Millipore, Billerica, MA); anti-KHSRP antibody (#Ab83291, Abcam, Toronto, ON, Canada); anti-HuR antibody (#A-21277, Molecular Probes courtesy of Life Technologies, Burlington, ON, Canada); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (AbD Serotec, Raleigh, NC); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA); HRP-conjugated anti-rabbit antibody (GE Healthcare Biosciences, Piscataway, NJ); enhanced chemiluminescent (ECL) substrate reagent (GE Healthcare Biosciences, Piscataway, NJ); Lowry DC protein assay (Biorad Laboratories, Mississauga, ON); recombinant CXCL8 protein (R & D Systems, Minneapolis, MN); siRNA targeting AUF-1 (S100300454 and S102653665), KHSRP (S100300587 and S100054691) and HuR (S100300139 and S103246551) (Qiagen, Toronto, ON, Canada); medium GC negative control non-targeting siRNA, OptiMEM reduced serum media and Lipofectamine RNAiMAX transfection reagent (Invitrogen, Burlington, ON, Canada).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Transfection, Control

Journal: Respiratory Research

Article Title: Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

doi: 10.1186/1465-9921-14-88

Figure Lengend Snippet: Effects of KHSRP-targeting siRNA on CXCL8 protein expression in HBE cells. HBE cells were treated for 24 h prior to harvesting cell supernatants for analysis with ELISA and whole cell lysates for analysis with immunoblotting. To determine the level of KHSRP protein knock-down membranes were probed with a specific KHSRP antibody, then were subsequently stripped and re-probed with an antibody to GAPDH to ensure equal protein loading (A ; representative of 3 separate experiments ) . Densitometry analysis with % knock-down relative to HRV+CSE alone is also shown (A ; n=3 ) . Cell supernatants were analyzed for CXCL8 protein (B ; n=5 ) . Data are presented as mean ± SEM. Lipid denotes transfection reagent alone. NT denotes non-targeting control siRNA. Asterisks denote a significant difference between the specified treatments (**p<0.01). ns = not significant.

Article Snippet: The following reagents were purchased from the indicated suppliers: bronchial epithelial cell basal medium and additives to create serum-free bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD); WI-38 cells and HRV type 16 (American Type Culture Collection, Manassas, VA); 3R4F research grade cigarettes (College of Agriculture Reference Cigarette Program, University of Kentucky) firefly luciferase reporter plasmid pGL4.10[ luc 2] and passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium Inc., Hayward, CA); TransIT-LT1 transfection reagent (Mirus, Madison, WI); anti-AUF-1 antibody (#07-260, Upstate curtsey of EMD Millipore, Billerica, MA); anti-KHSRP antibody (#Ab83291, Abcam, Toronto, ON, Canada); anti-HuR antibody (#A-21277, Molecular Probes courtesy of Life Technologies, Burlington, ON, Canada); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (AbD Serotec, Raleigh, NC); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA); HRP-conjugated anti-rabbit antibody (GE Healthcare Biosciences, Piscataway, NJ); enhanced chemiluminescent (ECL) substrate reagent (GE Healthcare Biosciences, Piscataway, NJ); Lowry DC protein assay (Biorad Laboratories, Mississauga, ON); recombinant CXCL8 protein (R & D Systems, Minneapolis, MN); siRNA targeting AUF-1 (S100300454 and S102653665), KHSRP (S100300587 and S100054691) and HuR (S100300139 and S103246551) (Qiagen, Toronto, ON, Canada); medium GC negative control non-targeting siRNA, OptiMEM reduced serum media and Lipofectamine RNAiMAX transfection reagent (Invitrogen, Burlington, ON, Canada).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Transfection, Control

Journal: Respiratory Research

Article Title: Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

doi: 10.1186/1465-9921-14-88

Figure Lengend Snippet: Effects of HuR-targeting siRNA on CXCL8 protein expression in HBE cells. HBE cells were treated for 24 h prior to harvesting cell supernatants for analysis with ELISA and whole cell lysates for analysis with immunoblotting. To determine the level of HuR protein knock-down membranes were probed with a specific HuR antibody, then were subsequently stripped and re-probed with an antibody to GAPDH to ensure equal protein loading (A ; representative of 3 separate experiments ) . Densitometry analysis with % knock-down relative to HRV+CSE alone is also shown (A ; n=3 ) . Cell supernatants were analyzed for CXCL8 protein (B ; n=6 ) . Data are presented as mean ± SEM. Lipid denotes transfection reagent alone. NT denotes non-targeting control siRNA. Asterisks denote a significant difference between the specified treatments (***p<0.001).

Article Snippet: The following reagents were purchased from the indicated suppliers: bronchial epithelial cell basal medium and additives to create serum-free bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD); WI-38 cells and HRV type 16 (American Type Culture Collection, Manassas, VA); 3R4F research grade cigarettes (College of Agriculture Reference Cigarette Program, University of Kentucky) firefly luciferase reporter plasmid pGL4.10[ luc 2] and passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium Inc., Hayward, CA); TransIT-LT1 transfection reagent (Mirus, Madison, WI); anti-AUF-1 antibody (#07-260, Upstate curtsey of EMD Millipore, Billerica, MA); anti-KHSRP antibody (#Ab83291, Abcam, Toronto, ON, Canada); anti-HuR antibody (#A-21277, Molecular Probes courtesy of Life Technologies, Burlington, ON, Canada); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (AbD Serotec, Raleigh, NC); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA); HRP-conjugated anti-rabbit antibody (GE Healthcare Biosciences, Piscataway, NJ); enhanced chemiluminescent (ECL) substrate reagent (GE Healthcare Biosciences, Piscataway, NJ); Lowry DC protein assay (Biorad Laboratories, Mississauga, ON); recombinant CXCL8 protein (R & D Systems, Minneapolis, MN); siRNA targeting AUF-1 (S100300454 and S102653665), KHSRP (S100300587 and S100054691) and HuR (S100300139 and S103246551) (Qiagen, Toronto, ON, Canada); medium GC negative control non-targeting siRNA, OptiMEM reduced serum media and Lipofectamine RNAiMAX transfection reagent (Invitrogen, Burlington, ON, Canada).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Knockdown, Transfection, Control

Journal: Respiratory Research

Article Title: Cigarette smoke enhances human rhinovirus-induced CXCL8 production via HuR-mediated mRNA stabilization in human airway epithelial cells

doi: 10.1186/1465-9921-14-88

Figure Lengend Snippet: HuR knockdown affects HRV+CSE-induced, but not basal, CSE or HRV-induced CXCL8 protein expression in HBE cells. HBE cells were treated for 24 h prior to harvesting cell supernatants for analysis with ELISA and whole cell lysates for analysis with immunoblotting. To determine the level of HuR protein knock-down membranes were probed with a specific HuR antibody, then were subsequently stripped and re-probed with an antibody to GAPDH to ensure equal protein loading (A ; representative of 3 separate experiments ) . Cell supernatants were analyzed for CXCL8 protein (B ; n=3 ) . Data are presented as mean ± SEM. Lipid denotes transfection reagent alone. NT denotes non-targeting control siRNA. Asterisks denote a significant difference between the specified treatments (*p≤0.05, **p<0.01 and ***p<0.001). ns = not significant.

Article Snippet: The following reagents were purchased from the indicated suppliers: bronchial epithelial cell basal medium and additives to create serum-free bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD); WI-38 cells and HRV type 16 (American Type Culture Collection, Manassas, VA); 3R4F research grade cigarettes (College of Agriculture Reference Cigarette Program, University of Kentucky) firefly luciferase reporter plasmid pGL4.10[ luc 2] and passive lysis buffer (Promega, Madison, WI); firefly luciferase assay kit (Biotium Inc., Hayward, CA); TransIT-LT1 transfection reagent (Mirus, Madison, WI); anti-AUF-1 antibody (#07-260, Upstate curtsey of EMD Millipore, Billerica, MA); anti-KHSRP antibody (#Ab83291, Abcam, Toronto, ON, Canada); anti-HuR antibody (#A-21277, Molecular Probes courtesy of Life Technologies, Burlington, ON, Canada); glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) antibody (AbD Serotec, Raleigh, NC); horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA); HRP-conjugated anti-rabbit antibody (GE Healthcare Biosciences, Piscataway, NJ); enhanced chemiluminescent (ECL) substrate reagent (GE Healthcare Biosciences, Piscataway, NJ); Lowry DC protein assay (Biorad Laboratories, Mississauga, ON); recombinant CXCL8 protein (R & D Systems, Minneapolis, MN); siRNA targeting AUF-1 (S100300454 and S102653665), KHSRP (S100300587 and S100054691) and HuR (S100300139 and S103246551) (Qiagen, Toronto, ON, Canada); medium GC negative control non-targeting siRNA, OptiMEM reduced serum media and Lipofectamine RNAiMAX transfection reagent (Invitrogen, Burlington, ON, Canada).

Techniques: Knockdown, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Transfection, Control