recombinant human del 1  (R&D Systems)

Journal: bioRxiv

Article Title: Melanoma cells release DEL-1 via small extracellular vesicles

doi: 10.1101/2022.04.04.487024

Figure Lengend Snippet: a) Left panel: Western Blot analysis for DEL-1 in whole cell lysates, culture supernatants and sEV isolated by a 2-step ultracentrifugation protocol in MCMDLN wildtype and DEL-1 overexpressing cells, recombinant DEL-1 is used as a positive control. Right panel: Western Blot analysis for DEL-1 in microvesicles collected after the 1 st ultracentrifugation step for 30 minutes at 18,000xg, and in sEV after the 2 nd ultracentrifugation step for 120 minutes at 100,000xg. The nature of the 52kD band in whole cell lysates remains open, but potentially corresponds to the predicted molecular size of 52kD of non-glycosylated DEL-1 (representative example of 3 repeats). “oex” denotes overexpression, “wt” denotes wild type b) Immunofluorescence of a GFP-tagged DEL-1 construct (green) transiently transfected into VM-14 melanoma cells and stained with EEA1 antibody (red). Co-localization is shown in yellow. Arrows indicate early endosomes. Insert shows a histogram of the quantified intensity profile between point A and B to confirm co-localization of DEL-1-GFP and EEA1 (representative example of 2 repeats). c) Size distribution of EEA1 positive/DEL-1 negative, EEA1/DEL-1 double positive intracellular structures as measured as area in µm 2 . d) Size distribution of DEL-1-GFP positive sEV isolated from cell culture supernatant by 2-step differential ultracentrifugation and analyzed by immunofluorescence analysis as measured as area in µm 2 . e) ECIS assay of a confluent endothelial monolayer treated with 10 µg/ml of recombinant DEL-1, 35 µg/ml RGD peptide, a combination thereof or PBS as a control. Transendothelial electrical resistance at 250 Hz was monitored for 2 hours and displayed as resistance change in ohms. Values are shown as mean ± SD. Statistical significance of the resistance change after 2 hours was calculated using one-way ANOVA; ns not significant, **** p <0.0001, n= ≧ 6.

Article Snippet: The predicted molecular size of DEL-1 is 52kDa; we detected a band at 65kDa, which corresponded to the size of recombinant DEL-1 expressed in Chinese hamster ovary cells (R&D Systems), indicating glycosylation.

Techniques: Western Blot, Isolation, Recombinant, Positive Control, Over Expression, Immunofluorescence, Construct, Transfection, Staining, Cell Culture

Journal: The Journal of Immunology Author Choice

Article Title: DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1

doi: 10.4049/jimmunol.1900746

Figure Lengend Snippet: DHEA restores TNF-mediated inhibition of DEL-1 expression in HUVEC. (A and B) HEK-293T cells (A) or HUVEC (B) were transfected with a hDEL-1-promoter-Luc reporter plasmid, followed by stimulation with or without TNF (10 ng/ml) for 16 h and analyzed for luciferase activity. Luciferase activity of untreated cells (−) was set as 1. Data are presented as mean ± SEM (n = 5 wells of HEK-293T or HUVEC cultures from one experiment representative of three). (C) HUVEC were stimulated with or without TNF (10 ng/ml) for 24 h, and DEL-1 mRNA expression was examined by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of untreated cells (−) was set in each experiment as 1. Data are presented as mean ± SEM (n = 4 independent experiments). (D) HUVEC were stimulated with or without TNF (10 ng/ml) for 6 h, and DEL-1 protein concentrations were determined in cell culture supernatants by ELISA. Data are presented as mean ± SEM (n = 4 independent experiments). (E) DEL-1 protein concentrations in cell culture supernatants of HUVEC transfected with siRNA against DEL-1 or nontargeting (ctrl) siRNA were measured by ELISA. Data are presented as mean ± SEM (n = 5 independent experiments). (F) HUVEC were transfected with a hDEL-1-promoter-Luc reporter plasmid, followed by pretreatment with DHEA (100 nM) or ethanol (ctrl) for 30 min and subsequent treatment with or without TNF (10 ng/ml) for 16 h. Luciferase activity of control vehicle–treated cells (ctrl) was set as 1. Data are presented as mean ± SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (G) HUVEC were pretreated for 30 min with increasing concentrations of DHEA or vehicle control (ethanol) at a concentration equal to that present in the highest DHEA dose (vehicle control groups are indicated as DHEA 0 nM), followed by stimulation with or without TNF (10 ng/ml) for 6 h. DEL-1 protein concentrations were determined in the cell culture supernatants by ELISA. Data are presented as mean ± SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (H) HUVEC were treated for 24 h with the indicated concentrations of DHEA or vehicle control (ethanol) at a concentration equal to that present in the highest DHEA dose (vehicle control group is indicated as DHEA 0 nM). DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA and relative gene expression of control vehicle–treated cells was set in each experiment as 1. Data are presented as mean ± SEM; n = 3. *p < 0.05.

Article Snippet: Detection of human DEL-1 protein in culture supernatants was performed by a sandwich ELISA, as described ( 21 ), using serial dilutions of recombinant human DEL-1 (R&D systems) for standard curve generation.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The Journal of Immunology Author Choice

Article Title: DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1

doi: 10.4049/jimmunol.1900746

Figure Lengend Snippet: TNF inhibits DEL-1 expression in a C/EBPβ–dependent manner. (A) HUVEC were transfected with a siRNA pool against C/EBPβ or a nontargeting siRNA pool, and C/EBPβ expression was examined by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of control siRNA-treated cells was set as 1. Data are presented as mean ± SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (B and C) HUVEC were transfected with a siRNA pool against C/EBPβ or a nontargeting siRNA pool and stimulated with or without TNF (10 ng/ml) for 6 h. (B) DEL-1 mRNA expression was analyzed by qPCR, using GAPDH mRNA for normalization. Relative gene expression of control siRNA-treated cells was set as 1. Data are presented as mean ± SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). (C) DEL-1 protein concentrations in cell culture supernatants were determined by ELISA. Data are presented as mean ± SEM (n = 5 wells of HUVEC cultures from one experiment representative of three). *p < 0.05.

Article Snippet: Detection of human DEL-1 protein in culture supernatants was performed by a sandwich ELISA, as described ( 21 ), using serial dilutions of recombinant human DEL-1 (R&D systems) for standard curve generation.

Techniques: Expressing, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1

doi: 10.4049/jimmunol.1900746

Figure Lengend Snippet: DHEA restores DEL-1 expression in TNF-treated HUVEC via the PI3K-AKT–C/EBPβ pathway. (A) HUVEC were treated with DHEA for indicated time points, and the phosphorylation status of AKT was examined by Western blot using vinculin as a loading control. One representative out of at least three experiments is shown. (B) HUVEC were preincubated for 1 h with the PI3K inhibitor LY294002 (20 μM) or its inactive analogue LY303511 (20 μM) or DMSO as control, followed by additional pretreatment with DHEA (100 nM) or ethanol as control for 30 min and subsequent stimulation with or without TNF (10 ng/ml) for 4 h. Chromatin was immunoprecipitated with nonimmune IgG or anti–C/EBPβ IgG and subjected to qPCR to amplify the −328- to −589-bp region of the DEL-1 promoter. Nonimmunoprecipitated cell extracts were used as input samples. Data are expressed as percentage of input. Means ± SEM are shown (n = 3 independent experiments). (C) HUVEC were preincubated for 1 h with the AKT inhibitor MK2206 (20 μM), PI3K inhibitor LY294002 (20 μM), or its inactive analogue LY303511 (20 μM) or DMSO as vehicle control, followed by additional pretreatment with DHEA (100 nM) or ethanol as vehicle control for 30 min and subsequent stimulation with or without TNF (10 ng/ml) for 4 h. DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of control vehicle–treated cells was set as 1. Data are presented as mean ± SEM (n = 5 wells of HUVEC cultures from one experiment, representative of three). *p < 0.05.

Article Snippet: Detection of human DEL-1 protein in culture supernatants was performed by a sandwich ELISA, as described ( 21 ), using serial dilutions of recombinant human DEL-1 (R&D systems) for standard curve generation.

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: The Journal of Immunology Author Choice

Article Title: DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1

doi: 10.4049/jimmunol.1900746

Figure Lengend Snippet: DHEA restores TNF-reduced DEL-1 expression via TRKA. (A) HUVEC were treated with DHEA for 5 min and phospho-TRKA levels were examined by Western blot using vinculin as a loading control. One representative out of at least three experiments is shown. (B) HEK-293T cells were cotransfected with a hDEL-1-promoter-Luc reporter plasmid and either a control vector (left panel) or TRKA cDNA plasmid (right panel). Cells were pretreated with ethanol (ctrl) or DHEA (100 nM) for 30 min followed by stimulation with or without TNF (10 ng/ml) for 16 h. Luciferase activity of control vehicle–treated cells was set as 1. Data are presented as mean ± SEM (n = 5 wells of HEK-293T or HEK-293TTRKA cultures from one experiment representative of three). (C) HUVEC were pretreated for 30 min with TRKA inhibitor (1 μM) or DMSO as vehicle control, followed by additional pretreatment with DHEA or ethanol as vehicle control for 30 min and subsequent stimulation with or without TNF (10 ng/ml) for 24 h, and DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of vehicle control–treated cells was set in each experiment as 1. Data are presented as mean ± SEM (n = 8 independent experiments). (D) HUVEC were pretreated with or without NGF (100 ng/ml) for 30 min, followed by stimulation with or without TNF (10 ng/ml) for 24 h. DEL-1 mRNA expression was analyzed by qPCR. Data were normalized to GAPDH mRNA, and relative gene expression of cells that were left untreated (ctrl) was set in each experiment as 1. Data are presented as mean ± SEM (n = 5 experiments). (E) HEK-293T cells were cotransfected with hDEL-1-promoter-Luc reporter plasmid and TRKA cDNA plasmid (HEK-293TTRKA). Cells were pretreated with or without NGF (100 ng/ml) for 30 min, followed by stimulation or not with TNF (10 ng/ml) for 16 h and analyzed for luciferase activity. Relative luciferase activity in untreated cells was set as 1. Data are presented as mean ± SEM (n = 5 wells of HEK-293TTRKA cultures from one experiment representative of three). *p < 0.05.

Article Snippet: Detection of human DEL-1 protein in culture supernatants was performed by a sandwich ELISA, as described ( 21 ), using serial dilutions of recombinant human DEL-1 (R&D systems) for standard curve generation.

Techniques: Expressing, Western Blot, Plasmid Preparation, Luciferase, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: DHEA Inhibits Leukocyte Recruitment through Regulation of the Integrin Antagonist DEL-1

doi: 10.4049/jimmunol.1900746

Figure Lengend Snippet: DHEA reduces neutrophil recruitment in an acute lung inflammation model in WT but not DEL-1–deficient mice. (A) WT and (B) DEL-1−/− mice were injected i.p. with DHEA or vehicle control solution (ctrl), followed by a second i.p. injection of DHEA or vehicle control solution after 24 h, as described in Materials and Methods. One and one-half hours later, mice received LPS intranasally, and after a further 24 h, neutrophil numbers were counted in the BAL fluid. Absolute neutrophil numbers are shown. Data are presented as mean ± SEM (n = 13 mice per group). *p < 0.05.

Article Snippet: Detection of human DEL-1 protein in culture supernatants was performed by a sandwich ELISA, as described ( 21 ), using serial dilutions of recombinant human DEL-1 (R&D systems) for standard curve generation.

Techniques: Injection