Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 1 Immunization with plasmid-encoded DLL4 results in an immunological response in BALB/c mice. (a) Expression of human DLL4 was verified by transient transfection of HeLa cells with empty vector or the vector encoding DLL4 (pDLL4) and analyzed by western blot. Recombinant human DLL4 was used as a positive control (rhDLL4). (b) Female BALB/c mice were immunized either intramuscularly (IM) or intradermally (ID) according to the depicted protocols. All intradermal injections were given in combination with electroporation (EP). One week after the final immunization animals were challenged by orthotopic injection of D2F2/E2 or TUBO cells in the mammary fat pad. (c) To investigate if the vaccination gave rise to an immunological response, serum was taken 10 days after the final immunization (protocol 1) and analyzed by human DLL4 enzyme-linked immunosorbent assay (ELISA). The figure depicts a representative analysis for one out of four mice analyzed.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Plasmid Preparation, Expressing, Transfection, Western Blot, Recombinant, Positive Control, Electroporation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 2 DNA vaccination against DLL4 results in suppression of tumor growth in BALB/c mice. (a) Expression of DLL4 in D2F2/ E2 tumor endothelial cells was assessed by immunofluorescent staining for the endothelial markers, CD31 (red) and DLL4 (green). Cell nuclei were counterstained by DAPI (blue). Growth of orthotopically implanted D2F2/E2 (b and c) or TUBO (d) mammary carcinomas in BALB/c mice following immunization with empty vector or with DLL4 plasmid DNA according to protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP; n ¼ 8 for all groups). The data shown in (b and d) are representative of three independent experiments. *Po0.05, **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Expressing, Staining, Plasmid Preparation

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 3 Targeting DLL4 by vaccination results in excessive formation of nonfunctional blood vessels. (a) Visual inspection of excised D2F2/E2 tumors revealed overt thrombosis in tumors from DLL4-vaccinated mice. (b) Blood vessel density of D2F2/E2 tumors visualized by immunostaining for the endothelial cell marker CD31 (red; n ¼ 5 for each group). (c) Perfused blood vessels of D2F2/E2 tumors as determined by vascular perfusion using fluorescein-labeled tomato lectin (green; n ¼ 5 for each group). The total area of the vascular bed was visualized by immunostaining for CD31 (red; n ¼ 5 for each group). The fraction perfused area was expressed as perfused area divided by total vascular area for each field. (d) Apoptotic index of D2F2/E2 tumor cells as determined by immunostaining for activated caspase-3 (n ¼ 5 for each group). *Po0.05, **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Immunostaining, Marker, Labeling

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 4 T cells are not mediators of the effector functions of the DLL4 vaccine. (a) Mouse DLL4-specific T cell responses following immunization by protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP) were assessed by IFN-g ELISpot by stimulation of splenocytes with mouse DLL4 peptides or recombinant mouse extracellular portion of DLL4 (n ¼ 5 for each group). Recombinant carcinoembryonic antigen protein and carcinoembryonic antigen peptides were used as negative controls. The polyclonal stimulant concanavalin A (conA) was used as a positive control. The experiment was repeated three times. (b) Splenocyte activity in mice following immunization by protocol 1 (3 ID þ EP) or protocol 2 (2 IM, 1 ID þ EP) at the time of sacrifice after tumor challenge was assessed by IFN-g ELISpot without antigen-specific stimulation (vector, n ¼ 9; protocol 1, n ¼ 8; protocol 2, n ¼ 5). (c) Depletion of CD4 þ or CD8 þ cells was initiated three days before tumor challenge by injection of anti-CD4 or anti-CD8 antibodies and verified by flow cytometric analysis of peripheral blood 5 days after injection of antibodies. Tumor growth curve from vector-immunized mice was duplicated from Figure 2c, which depicts an experiment performed simultaneously to the T cell depletion experiment. (d) Growth of orthotopically implanted D2F2/E2 tumors in control mice and in mice depleted of CD4 þ/CD8 þ cells following vaccination with DLL4 plasmid DNA (n ¼ 6 for all groups). **Po0.01, ***Po0.001; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Enzyme-linked Immunospot, Recombinant, Positive Control, Activity Assay, Plasmid Preparation, Injection, Control

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 5 Tumor growth suppression in DLL4-vaccinated mice is mediated by induction of anti-DLL4 antibodies. (a) The presence of anti-mouse DLL4 antibodies in the serum of DLL4-immunized mice was analyzed by ELISA. The figure depicts a representative analysis for one out of four mice analyzed. (b) IsolectinB4 staining of wholemounted P5 mouse retinas from mice treated with immune serum from vector or DLL4 immunized mice according to protocol 1 (3 ID þ EP; n ¼ 4 for each group). Arrowheads indicate endothelial cells morphologically identified as tip cells. (c) Growth of orthotopically implanted D2F2/E2 tumors in mice treated with serum from mice immunized with vector (n ¼ 12) or DLL4 plasmid DNA (n ¼ 16) according to protocol 1 (3 ID þ EP). The experiment was repeated twice with similar results. **Po0.01; Student’s t-test.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Plasmid Preparation

Journal: Oncogene

Article Title: Therapeutic efficacy of a DNA vaccine targeting the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma.

doi: 10.1038/onc.2010.176

Figure Lengend Snippet: Figure 6 No evidence for liver toxicity or delayed wound healing following immunization with the DLL4 vaccine. (a) Hematoxylin and eosin staining of tissue sections of liver from mice that initiated the immunization procedure with empty vector or the DLL4 vaccine 5 months earlier. (b) Rate of healing expressed as % closure of wounds inflicted to mice immunized with empty vector or the DLL4 vaccine (n ¼ 8 mice per group, two wounds per mouse). *Po0.05, repeated-measures ANOVA.

Article Snippet: Plates were coated overnight at 4 1C with 2 pmol of recombinant human DLL4 (Bioinvent International, Lund, Sweden) or 1 pmol of recombinant mouse DLL4 (R&D Systems) per well in 50ml of 0.1M sodium carbonate, pH 9.5.

Techniques: Staining, Plasmid Preparation

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Inhibition assay of sCD4 and CD4-derived protein molecules against pseudotyped and infectious Ebola viruses (A) sCD4 (2D), soluble first two-domain CD4; sCD4 (4D), soluble four-domain CD4; CD4-Ig, first two-domain CD4 linked with human Fc region (Ig domain) ; CD4-IgG2, first two-domain CD4 with human Fc region (Ig domain); EBOV, Zaire Ebola virus; BDBV, Bundibugyo ebolavirus. All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments. (B) sCD4 (4D) inhibits infectious Ebola virus infection. This experiment was conducted in BSL-4 containment in triplicates, and the error bars represent standard deviations.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Inhibition, Derivative Assay, Virus, Infection

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Inhibition assay of CD4-mimetic small molecules against pseudotyped Ebola viruses (A) NBD-556 and analogs inhibiting EBOV infection. (B) Newly designed and synthesized compounds with phenyl ring substituted molecules in the region-I inhibiting EBOV. All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Inhibition, Infection, Synthesized

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Specificity assay of CD4-IgG-2 and JRC-II-191 against pseudo-typed HIV, EBOV, VSV, and A-MLV CD4-mimetic molecules CD4-IgG2 and JRC-II-191 were evaluated against four different viruses: HIV (YU2), EBOV, VSV (vesicular stomatitis virus), and A-MLV (amphitropic murine leukemia virus). All experiments were conducted in triplicates and the error bars represent standard deviations from three experiments.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Virus

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Binding affinity and kinetics assay of sCD4 (2D) and NBD-556 binding to EBOV receptor binding domain by biolayer interferometry Two-domain CD4 (sCD4-2D) (A) and NBD-556 (B) at two pH conditions, pH7.4 and pH6.1. Open circle in (B) was not included in the fit of the data. (See the BLI section for details).

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Binding Assay

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Binding competition assay of sCD4 and CD4-mimetic compounds with receptor NPC1 (A) sCD4, NBD-556, JRC-II-191, and DY-III-228 competing with NPC1 receptor. (B) Dose response (0, 10, 20, and 40 μM) of NBD-556 in binding competition with NPC1 receptor. (C) Comparison of wild-type (EGPDCM-WT) and mutant (EGPDCM-mut) (WF/AA, 86W/A, and 88F/A) receptor binding domain (RBD) of EBOV binding to the NPC1 receptor. The mutant (WF/AA) surface expression level was confirmed to be comparable to the wild type (see ). Representative of 3 experiments.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Binding Assay, Competitive Binding Assay, Comparison, Mutagenesis, Expressing

Journal: iScience

Article Title: Soluble CD4 inhibits Ebola virus infection by targeting endosomal receptor-binding site

doi: 10.1016/j.isci.2025.112573

Figure Lengend Snippet: Molecular docking analysis of sCD4 and CD4mcs (A) Molecular docking analysis of sCD4 and CD4mcs binding to the EBOV-GP. sCD4 docking using HDOCK program. The docking energy is −150.41 kcal/mol. CD4mcs docking using AutoDock program. (a) sCD4-RBD ribbon model; (b) sCD4-RBD surface binding model; (c) sCD4-RBD interactions: hydrogen bond: GP T83:OG1-CD4 L44:N (green) and salt bridge: GP K84:NZ-CD4 D56:OD2 (brown) ; (d) NBD-556 docking ribbon model (−6.477 kcal/mol); (e) NBD-556 docking surface model; and (f), superimposed of NBD-556 (yellow), JRC-II-191 (magenta), and DY-III-228 (cyan). (B) Comparisons of two receptor binding sites of CD4bs and NPC1-bs. (a) CD4bs , of HIV gp120 (based on PDB 1G9N , YU2 strain). (b) NPC1bs , of EBOV-GP (based on PDB 5F1B , Zaire EBOV). Showing the accommodation of CD4-mimetic compound NBD-556 in the red dash circle. Hydrophobic surface in gray; CD4bs of HIV gp120 in magenta dash circle; NPC1bs of EBOV-GP in red dash circle.

Article Snippet: Human soluble CD4 (4D) protein (CD4-3167H) was also purchased from Creative Biomart Inc. CD4-Ig Recombinant protein was obtained from NIH HIV Reagent Program.

Techniques: Binding Assay