quantikine elisa human cxcl5 ena 78 immunoassay kit  (R&D Systems)

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Modulation of CXCL5 in the cell proliferation and invasion of prostate cancer cells. ( A ) CXCL5 mRNA levels of prostate cells were determined by RT-qPCR. Data are presented as the ΔCT relative to β-actin. The cell immunofluorescent staining of CXCR2 protein in LNCaP ( B ) and PC-3 ( C ) cells was determined by flow cytometry. Data from quantitative analysis represented the percentage of CXCR2-positive cells. ( D ) CXCL5 levels in the supernatant from PC_shCOL and PC_shCXCL5 cells were assessed by ELISA. ( E ) The mRNA levels (±SE, n = 3) of CXCL5 and HO-1 of PC_shCOL cells relative to PC_shCXCL5 cells. ( F ) Reporter activity (±SE, n = 6) of HO-1 reporter vector after co-transfected with various dosages of CXCL5 expression vectors. ( G ) The protein levels of the CXCR2, CXCL5, and HO-1 of mock-transducted PC-3 (PC_shCOL) and CXCL5 knockdown PC-3 (PC_shCXCL5) cells were examined by immunoblot assays. Quantitative analysis (±SE, n = 3) was presented as a relative density of proteins/β-actin. ( H ) The abilities of cellular proliferation in PC_shCOL and PC_shCXCL5 cells were measured by flow cytometry using a Ki67 flow cytometry kit (±SE, n = 3). ( I ) The protein levels of EMT markers (E-cadherin, N-cadherin, Snail, Slug, and Vimentin) in PC_shCOL and PC_shCXCL5 cells were determined by immunoblot assays and quantitative analysis (±SE, n = 3). ( J ) The cellular invasion ability was determined by in vitro Matrigel invasion assays. Data are presented as the mean percentage (±SE; n = 3) in relation to the PC_shCOL cells. ( K ) The cell numbers for neutrophil trans-membrane migration induced by supernatant from PC_shCOL and PC_shCXCL5 cells (±SE, n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Quantitative RT-PCR, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Plasmid Preparation, Transfection, Expressing, Knockdown, Western Blot, In Vitro, Membrane, Migration

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Knockdown of CXCL5 blocks PC-3 cell tumor growth of cells in xenograft mouse models. Athymic male nude mice were subcutaneously injected with PC_shCOL or PC_shCXCL5 cells for 33 days. ( A ) Photographs of representative xenografted mice and tumors. ( B ) The tumor sizes derived from PC_shCOL and PC_shCXCL5 were measured every 3 days. ( C ) Average body weights (mean ± SE) of mice during the experimental period. ( D ) Quantitative data (mean ± SE; n = 6) describing tumor weight of the PC_shCOL and PC_shCXCL5 groups when the mice were sacrificed on day 33. ( E ) Whole-cell lysates of tumor samples from the PC_shCOL and PC_shCXCL5 groups were subjected to immunoblot assays for CXCL5, HO-1, and β-actin. ( F ) The mRNA levels of CXCL5 and HO-1 in the xenografted tumors were analyzed using RT-qPCR assays (±SE, n = 3). ** p < 0.01.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Knockdown, Injection, Derivative Assay, Western Blot, Quantitative RT-PCR

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Modulation of CXCL5 and SB225002 in endogenous and H 2 O 2 -induced ROS in prostate cancer PC-3 cells. ( A ) ROS levels and quantitative data from PC_shCOL and PC_shCXCL5 cells after treatment with or without H 2 O 2 , as measured by flow cytometry. ( B ) The levels of the CXCR2, HO-1, and CXCL5 proteins after treatment with various concentrations of SB225002, as indicated, as examined by immunoblotting assays. Quantitative data are presented as the intensity of the protein bands of the target proteins/β-actin relative to the vehicle-treated group. ( C ) ROS levels and quantitative data from PC3 cells after treatment with various concentrations of SB225002 and with/without H 2 O 2 , as measured by flow cytometry. ** p < 0.01.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Flow Cytometry, Western Blot

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Modulation of CXCL5 in neutrophil migration and cell proliferation in prostate cancer LNCaP cells. ( A ) Protein levels of CXCL5 and HO-1 from mock-transfected LNCaP (LN-DNA) and LNCaP-overexpressed CXCL5 (LN-CXCL5) cells were examined by immunoblot assays. Quantitative analysis (±SE, n = 3) is presented as the relative density of target proteins/β-actin. ( B ) The mRNA levels (±SE, n = 3) of CXCL5 and the HO-1 of LN-DNA and LN-CXCL5 cells, as determined by RT-qPCR. ( C ) CXCL5 levels in the supernatant of LN-DNA and LN-CXCL5 cells, as evaluated by ELISA. ( D ) The numbers of neutrophil transmembrane migration cells induced by supernatant from LN-DNA and LN-CXCL5 cells (±SE, n = 3). ( E ) The cellular proliferation abilities of LN-CXCL5 relative to LN-DNA cells were measured by flow cytometry using a Ki67 flow cytometry kit (±SE, n = 3). ( F ) Cell cycle analysis of LN-DNA and LN-CXCL5 cells. ( G ) Cell cycle modulators’ protein levels were determined by immunoblot assays and quantitative analysis (±SE, n = 3). * p < 0.05, ** p < 0.01. ND: not detectable.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Migration, Transfection, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Cycle Assay

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Modulation of CXCL5 via CXC2R in endogenous and H 2 O 2 -induced ROS in prostate cancer LNCaP cells. ( A ) The protein levels of CXCR2, PSA, and HO-1 of LN-DNA, LN-CXCL5, and SB225002-treated LN-CXCL5 cells were examined by immunoblot assays. Quantitative analysis (±SE, n = 3) is presented as the relative density of target proteins/β-actin. ( B ) Reporter activity (±SE, n = 6) of the PSA reporter vector after co-transfected with various doses of CXCL5 expression vectors. ( C ) PSA levels (±SE, n = 6) in the supernatant of LN-DNA, LN-CXCL5, and LN-CXC5 treated with CXCL5 antibody or CXCR2 antibody, as assessed by ELISA. Data are presented as PSA secretion in relation to the LN-DNA group. ( D ) ROS levels and quantitative data (±SE, n = 3) of LN-DNA and LN-CXCL5 cells after treatment with or without H 2 O 2 and SB225002, as measured by flow cytometry. ( E ) HO-1 protein levels when LN-CXCL5 cells were transiently knocked down as to the HO-1 gene by immunoblot assays and quantitative analysis (±SE, n = 3). ( F ) ROS levels and quantitative data (±SE, n = 3) from LN-DNA, LN-CXCL5, and HO-1-knockdown LN-CXCL5 cells after treatment with/without H 2 O 2 , as measured by flow cytometry. ** p < 0.01. N.S., no significant difference.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Western Blot, Activity Assay, Plasmid Preparation, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Knockdown

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Modulation of CXCL5 in cell contraction and migration in prostate stroma myofibroblast WPMY-1 cells. ( A ) Cell immunofluorescent staining of the CXCR2 protein in WPMY-1 cells was determined by flow cytometry. The data from the quantitative analysis represents the percentage of CXCR2-positive cells. ( B ) Protein levels of Fibronectin, α-SMA, HO-1, CXCL5, and β-actin in WPMY-1-DNA and WPMY-1-CXCL5 cells, as determined by immunoblot assays and quantitative analysis (±SE, n = 3). ( C ) Cell contraction of mock-transfected WPMY-1 (WPMY-1-DNA) and CXCL5-overexpressed WPMY-1 (WPMY-1-CXCL5) cells, as measured by collagen contraction assays. Data are presented as the mean percentage (±SE; n = 3) of WPMY-1-CXCL5 cells in relation to WPMY-1-DNA cells. ( D ) Cell contraction of WPMY-1 cells when treated with the supernatant from the LN-DNA, LN-CXCL5, or LN-CXCL5 with SB225002. Data are presented as the mean percentage (±SE; n = 3) in relation to the supernatant from LN-DNA-treated WPMY-1 cells. ( E ) Migration capabilities in WPMY1 cells treated with conditioned media of LN-DNA, LN-CXCL5, or LN-CXCL5 with SB225002. The white line indicates the average of the leading edges of cells and the size of the wound area. Data are presented as the mean percentage (±SE; n = 3) in relation to the supernatant from LN-DNA-treated WPMY-1 cells. * p < 0.05, ** p < 0.01.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Migration, Staining, Flow Cytometry, Western Blot, Transfection

Journal: Antioxidants

Article Title: The C-X-C Motif Chemokine Ligand 5, Which Exerts an Antioxidant Role by Inducing HO-1 Expression, Is C-X-C Motif Chemokine Receptor 2-Dependent in Human Prostate Stroma and Cancer Cells

doi: 10.3390/antiox13121489

Figure Lengend Snippet: Modulation of CXCL5 and SB225002 in endogenous and H 2 O 2 -induced ROS in prostate stroma myofibroblast WPMY-1 cells. ( A ) The protein levels of α-SMA, CXCR2, and HO-1 of WPMY-1 cells after treatment with the conditioned media of the LN-DNA, LN-CXCL5, or LN-CXCL5 with SB225002, as examined by immunoblot assays. Quantitative analysis (±SE, n = 3) is presented as the relative density of target proteins/β-actin. ( B ) ROS levels and quantitative data for WPMY-1 cells after treatment with the conditioned media of the LN-DNA, LN-CXCL5, or LN-CXCL5 with SB225002, and with/without H2O2, as measured by flow cytometry. ( C ) ROS levels and quantitative data from WPMY-1-DNA, WPMY-1-CXCL5, and SB225002-treated WPMY-1-CXCL5 cells, after treatment with or without H 2 O 2 , as measured by flow cytometry. ( D ) ROS levels and quantitative data for WPMY-1 cells after treatment with 2 μM SB225002 and with/without H 2 O 2 , as measured by flow cytometry. * p < 0.05, ** p < 0.01.

Article Snippet: After incubation with 1 mL of RPMI 1640 medium with 10% FCS for 24 h, the conditioned media of PC_shCOL, PC_shCXCL5, LN-DNA, and LN-CXCL5 cells were collected for CXCL5 assays using a Quantikine ELISA human CXCL5/ENA-78 immunoassay kit, conducted as described by the manufacturer (Catalog #DX000; R&D Systems Inc.).

Techniques: Western Blot, Flow Cytometry