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: International journal of molecular sciences

Article Title: Experimental Evaluation of Quantum Dots and Antibodies Conjugation by Surface Plasmon Resonance Spectroscopy.

doi: 10.3390/ijms232012626

Figure Lengend Snippet: Figure 4. Dependence of Au/CD44/anti-CD44 complex dissociation and surface regeneration efficiency on the nature of the regeneration solution.

Article Snippet: Recombinant human CD44 biomarker (career free, His tag C-Termus), monoclonal mouse IgG clone # 2C5 (anti-CD44) and recombinant human growth hormone from E. coli (hGH) were obtained from R&D Systems (Abingdon, UK).

Techniques:

Journal: Cellular immunology

Article Title: In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade.

doi: 10.1016/j.cellimm.2025.104932

Figure Lengend Snippet: Fig. 2. Tumor conditioning induces macrophage CD163 and IL-10 gene expression. Tumor-conditioned human monocyte-derived macrophages (M(A2780)) were generated and their gene expression was compared to control macrophages by RT-qPCR. A-F: Presents gene expression of polarization markers (CD163, IL-10, and TNF-α) and phagocytosis checkpoints (SIRPα, LILRB1, and Siglec-10) in tumor-associated macrophages compared to controls. Statistics: comparisons were made against controls using paired t-tests and a significance level of p = 0.05. When indicated by a logarithmic y-axis, comparisons were made on log-transformed data. Data are presented as individual data points with mean ± SD. N = 4.

Article Snippet: The plates were read at 450/ 620 nm and a standard curve ranging from 0.625 to 8 μg/L was prepared using recombinant human LILRB1 (R&D systems, 8989-T2). sCD206 (samples diluted 1:5) [34], and sSIRPα (samples diluted 1:10) [35] were measured as previously reported in detail.

Techniques: Gene Expression, Derivative Assay, Generated, Control, Quantitative RT-PCR, Transformation Assay

Journal: Cellular immunology

Article Title: In vitro ovarian tumor-conditioned CD163+ human macrophages retain phagocytic response to CD47 blockade.

doi: 10.1016/j.cellimm.2025.104932

Figure Lengend Snippet: Fig. 3. Tumor cell conditioning induces CD163, CD206, CD80 and LILRB1 on the membrane protein level. Tumor-conditioned human monocyte-derived macrophages (M(A2780)) were generated and their membrane protein expression compared to control macrophages by flow cytometry. The gating strategy is presented in Supplementary fig. 1. A-G: Presents median fluorescence intensities (MFI) of polarization markers (CD163, CD206, and CD80) and MFI values and percent macrophages positive for phagocytosis checkpoints (LILRB1, and Siglec-10). Statistics: comparisons were made against controls using paired t-tests and a significance level of p = 0.05. When indicated by a logarithmic y-axis, comparisons were made on log-transformed data. Data are presented as individual data points with mean ± SD. N = 9.

Article Snippet: The plates were read at 450/ 620 nm and a standard curve ranging from 0.625 to 8 μg/L was prepared using recombinant human LILRB1 (R&D systems, 8989-T2). sCD206 (samples diluted 1:5) [34], and sSIRPα (samples diluted 1:10) [35] were measured as previously reported in detail.

Techniques: Membrane, Derivative Assay, Generated, Expressing, Control, Flow Cytometry, Fluorescence, Transformation Assay

Journal: Cell reports

Article Title: Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5

doi: 10.1016/j.celrep.2023.113145

Figure Lengend Snippet:

Article Snippet: MYC Epitope Tag Antibody Rabbit Polyclonal , Rockland , Cat#600-401-381; RRID:AB_217927.

Techniques: Control, Recombinant, Affinity Purification, Virus, Plasmid Preparation, Quantitative Proteomics, Over Expression, Knock-Out, Software

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: (A) ArcA phosphorylation. V. cholerae overexpressing N-terminally His-tagged ArcA WT or ArcA D54A were grown aerobically (O 2 + ), microaerobically (O 2 − ), or microaerobically in the presence of 50 μM CHP. Whole-cell lysates were analyzed by Phos-Tag gel electrophoresis followed by anti-His6 western blotting to visualize ArcA phosphorylation. (B) ROS effects on ArcA repression of sdhC transcription. Wild type, Δ arcA , and arcA C173S mutants containing P sdhc -lacZ reporter plasmids were grown in AKI medium to mid-log phase and treated with or without 60 μM CHP for 1 h. β-Galactosidase activity was measured and normalized against viable cell numbers (SU, special units = β-gal unit/CFU × 10 6 ). The mean of nine datapoints from three independent assays is shown, and error bars represent the standard deviation; ****, p < 0.0001 (Student’s t test); ns, no significance. (C) EMSA assays. Purified ArcA wild type and ArcA-mutant derivative proteins were treated with DTT (R, reduced), carbamoyl phosphate (P, phosphorylated), and CHP (O, oxidized) before incubating with the 32 P-labeled sdhC promoter DNA. The reaction mixes were separated on a native gel and imaged on a Typhoon phosphorimager.

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Phospho-proteomics, Nucleic Acid Electrophoresis, Western Blot, Activity Assay, Standard Deviation, Purification, Mutagenesis, Labeling

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: (A and B) ArcA-ArcA interaction. E. coli BTH101 containing pKT25- arcA and pUC18c- arcA and derivatives were grown for 12 h at 37°C in LB aerobically or microaerobically (A) or microaerobically with or without 50 μM CHP (B). Single-step β-galactosidase activity assays were performed, and the LacZ unit was calculated by the formula described in . The mean of nine datapoints from three independent assays is shown, and error bars represent the standard deviation; ***, p < 0.001 (Student’s t test); ns, no significance. (C) V. cholerae containing overexpressed His6-tagged ArcA variants were grown under the virulence-inducing condition. When indicated, 50 μM CHP was added during ArcA induction. Cell lysates (normalized by optical density at 600 nm [OD 600 ]) with or without 100 mM BME were separated on a non-reducing 12% SDS-polyacrylamide gel followed by western blotting analysis using anti-His6 antibody; O-M, oxidized monomer; R-M, reduced monomer; O-D, oxidized dimer. (D) Full MS spectrum of the [M + 3H] 3+ ( m/z = 675.30) ions of the disulfide-linked ALLHFC 173 ENPGK and FC 233 GDLED. (E) Working model. ArcA in the reduced unphosphorylated state is in an inactive form as monomers. A microaerobic environment leads ArcA phosphorylation and thus ArcA-ArcA interaction, allowing target DNA binding. When cells are challenged by ROS, ArcA phosphorylation is abolished, but an intramolecular disulfide bond is formed, introducing a conformational change that retains ArcA-ArcA interaction and DNA binding.

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Activity Assay, Standard Deviation, Western Blot, Phospho-proteomics, Binding Assay

Journal: Cell reports

Article Title: Thiol-based functional mimicry of phosphorylation of the two-component system response regulator ArcA promotes pathogenesis in enteric pathogens

doi: 10.1016/j.celrep.2021.110147

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-His6 antibody , Rockland Immunochemicals , Cat# 200-301-382: RRID: AB_10703081.

Techniques: Virus, Expressing, Plasmid Preparation, Recombinant, cDNA Synthesis, Control, Software, Sequencing