Journal:

Article Title: CTLA-4 regulates allergen response by modulating GATA-3 protein level per cell

doi: 10.1111/j.1365-2567.2007.02537.x

Figure Lengend Snippet: Effects of in vitro CTLA-4 blockade on GATA-3 protein level/cell and frequency of IL-4-producing cells. Naive CD4+ T cells purified from the spleens of normal mice were induced to differentiate with anti-CD3 mAb, a recombinant form of CD80-Fc, IL-4 and anti-IFN-γ mAb for 3 days. Anti-CTLA-4 mAb was added in soluble form at increasing concentrations (0·1–10 μg/ml). (a) Mean fluorescence intensity for GATA-3 staining, (b) percentage of IL-4+ cells.

Article Snippet: 18 CD4 cell culture For in vitro experiments, splenic naive CD4 cells were purified from non-immunized BALB/c mice by immuno-magnetic cell sorting as previously described 19 and cultured in 24-well plates precoated with anti-CD3ε mAb (clone 145-2C11; 10 μg/ml) and/or recombinant mouse B7-1(CD80)/Fc chimeric protein (R & D Systems, cat. 740-B1; 0·33 μg/ml).

Techniques: In Vitro, Purification, Recombinant, Fluorescence, Staining

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: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Effect of LacCer on the enzymatic activity of recombinant human cPLA2 in a cell-free assay. (unpaired T test, compared to control) (B) Pla2g4a expression in astrocytes 51 days after NOD EAE induction, and PLA2G4A expression in CNS samples from MS patients and controls (The same set of samples as Figure 1C. (unpaired T test compared to Naïve or Control) (C) EAE development in NOD mice treated 39 and 47 days after EAE induction with lentiviral constructs expressing control or Pla2g4a targeting shRNAs in astrocytes. (n=7, N=2, Regression slope T test, compared to shControl) (D-G) CNS samples were harvested 51 days after EAE induction from mice treated with lentivirus-delivered shRNAs shown in Figure 2C. (unpaired T test, compared to shControl) (D) Axonal loss and demyelination in spinal cord. (E) the number CNS-infiltrating inflammatory monocytes. (F) mRNA expression in astrocytes determined by qPCR. (G) Whole genome expression in astrocytes from control or Pla2g4a knockdown mice (n=3) (H,I) Astrocyte conditioned medium was prepared for test in in vitro monocyte migration (unpaired T test compared to activated shControl group) (H) and neurotoxicity assays (I). Migrating monocytes and neuronal death in the resting shControl-treated group were set as 100%. (J) Pla2g4a was knocked down in astrocytes, which were then activated, and mRNA expression was determined by qPCR in microglia co-cultured with the astrocytes. (unpaired T test, compared to shControl) (K) The effect of LacCer on cPLA2 enzymatic activity in astrocytes. (unpaired T test, compared to all) (L) mRNA expression determined by qPCR in activated astrocytes in the presence of cPLA2i. (unpaired T test, compared to all) (M) mRNA expression determined by qPCR in human astrocytes activated in the presence of LacCer, cPLA2i or both. (unpaired T test, compared to all) (N) Effect of cPLA2 inhibition on NF-κB activation in astrocytes. The nucleus p65 level is analyzed by western blot. (unpaired T test, compared to all) (O) Effect of cPLA2 inhibition determined by ChIP assay on NF-κB recruitment to responsive elements in Ccl2, Csf2 and Nos2 promoters. (unpaired T test, compared to all)

Article Snippet: Mutagenesis and Cloning To generate expression plasmids for mutant or truncated overexpression plasmids, human PLA2G4A-His tag overexpression plasmid (#MG51714-CH, Sino Biological) and human MAVS-flag overexpression plasmid (#45905, Addgene) were used as templates.

Techniques: Activity Assay, Recombinant, Cell-Free Assay, Expressing, Construct, In Vitro, Migration, Cell Culture, Inhibition, Activation Assay, Western Blot

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Effect of LacCer and cPLA2 inhibition on MAVS oligomerization, analyzed by SDD-AGE and western blot. (unpaired T test, compared to all, intensity normalized to vehicle Naïve condition) (B) ROS levels in activated murine and data are shown relative to ROS levels in resting astrocytes. (unpaired T test, compared to all) (C) LacCer induces cPLA2 recruitment to mitochondria and co-localization with MAVS. Astrocytes were treated with 10 μM LacCer or vehicle for 4 hours then stained and analyzed by confocal microscopy. Bar plots depict the ratio of total cPLA2 co-localization with mitochondria, the ratio of total MAVS co-localization with mitochondria, and the ratio of cPLA2-MAVS co-localization in mitochondria. (n≥25 cells per group, unpaired T test, compared to vehicle) (D) MAVS-cPLA2 interaction in astrocyte is analyzed by CoIP assay with MAVS-specific antibody and western blot. (unpaired T test, compared to all, intensity normalized to vehicle naive condition) (E,F) Domain binding analysis for cPLA2-MAVS interaction. Full-length human cPLA2 together with flag-tagged human MAVS (Full length, W56A mutant, G67A/W68A/V69A mutant (AAA), M2–6L mutant or M2 isoform) (E); or human cPLA2 (full-length, phospholipid binding domain deficient or C2 domain deficient) together with full-length flag-tagged human MAVS (F) were co-expressed in HEK293 cells, protein complexes were pulled down with anti-Flag antibodies and analyzed by western blot. (G) The effect of cPLA2 overexpression in trigger MAVS oligomerization. cPLA2 (full-length and C2 domain only) and MAVS were co-expressed in HEK293 cells, mitochondria were isolated 24 hours later and analyzed by SDD-AGE and western blot. (H) The effect of cPLA2 overexpression in triggering MAVS-mediated NF-κB activation, analyzed by luciferase assay. (unpaired T test, compared to all) (I) Effect of MAVS on NF-κB activation in WT and Mavs knockout astrocytes. The level of nucleus p65 level is analyzed by western blot. (unpaired T test, compared to corresponding condition between WT and MAVS−/− astrocytes) (J) mRNA expression determined by qPCR in activated WT and MAVS−/− astrocytes. (unpaired T test, compared to corresponding condition between WT and MAVS−/− astrocytes) (K) Recruitment of NF-κB to Ccl2, Csf2 and Nos2 promoters in murine WT and MAVS−/− astrocytes in culture determined by ChIP assay as in Figure 2O. (unpaired T test, compared to all) (L) EAE development in NOD mice treated with lentiviral constructs expressing shRNAs targeting Mavs or control in astrocytes 37 and 43 days after EAE induction. (n≥7, N=3, Regression slope T test, compared to shControl). (M,N) CNS samples were harvested 50 days after EAE induction from mice treated with lentivirus-delivered shRNAs shown in Figure 3L. (unpaired T test, compared to shControl) (M) The number of CNS-infiltrating inflammatory monocytes. (N) Inflammatory genes expression in astrocytes from control and Mavs knockdown mice was detected by nCounter analysis system (n=4). (O, P) Astrocyte-conditioned medium was prepared from activated WT or MAVS−/− astrocytes for test in in vitro monocyte migration. (O, unpaired T test, compared to all) and neurotoxicity assays (P, unpaired T test, compared to all). Migrating monocytes and neuronal death in the resting shControl-treated group were set as 100%.

Article Snippet: Mutagenesis and Cloning To generate expression plasmids for mutant or truncated overexpression plasmids, human PLA2G4A-His tag overexpression plasmid (#MG51714-CH, Sino Biological) and human MAVS-flag overexpression plasmid (#45905, Addgene) were used as templates.

Techniques: Inhibition, Western Blot, Staining, Confocal Microscopy, Co-Immunoprecipitation Assay, Binding Assay, Mutagenesis, Over Expression, Isolation, Activation Assay, Luciferase, Knock-Out, Expressing, Construct, In Vitro, Migration

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Level change of glucose metabolism in activated astrocytes measured by metabolomic profiling. Green color indicates metabolites decreased by cPLA2i treatment, red color indicates increased metabolites, black color indicates metabolites for which no data were collected. (B, C) Effect of cPLA2 (cPLA2i) or MPCs (UK-5099) inhibition in astrocyte on lactate release (B, unpaired T test, compared to activated vehicle condition, normalized to naive vehicle condition) and pyruvate level in mitochondria (C, unpaired T test, compared to activated vehicle condition, normalized to naive vehicle condition). (D,E) Metabolomic profiling analysis of activated astrocytes in the presence of cPLA2i. (D) Effect of cPLA2 inhibition on saturated and unsaturated fatty acids level in activated astrocyte, shown relative to their levels on resting astrocytes. Each dot indicates a different fatty acid. (unpaired T test, compared to activated vehicle condition) (E) Metabolite categories altered by cPLA2i treatment. (F) Effect of methylglyoxal supply on lactate release by activated astrocytes. (unpaired T test, compared to activated control condition) (G) Effect of cPLA2 or MPCs inhibition in mitochondrial function of astrocyte analyzed by Mito Stress test assay. (unpaired T test, compared to all) (H,I) Effect of gene targeted siRNA knockdown in mitochondrial function of LacCer loaded astrocytes analyzed by Mito Stress test assay (H, unpaired T test, compared to all) and lactate release by astrocytes (I, unpaired T test, compared to naive siControl condition). (J) Effect of LacCer loading or cytokines stimulation on the interaction of MAVS with HK2 and cPLA2 in astrocyte, evaluated by pull-down assay and western blot. (K) Mitochondrial HK enzymatic activity in activated astrocytes. (unpaired T test, compared to vehicle condition) (L) MAVS binding domain analysis. Flag-tagged human MAVS (Full length, M2–6L, M2, AAA or W56A) was over-expressed in HEK293 cell and 24 hours later MAVS protein complexes were pulled down and analyzed by western blot. (M) Human cPLA2 (cPLA2-GFP or C2 domain-GFP respectively) were over-expressed in HEK293 cell and 24 hours later MAVS protein complexes were pulled down and analyzed by western blot. (N) Effect of cPLA2 or cPLA2 C2 domain overexpression in HEK293 cells on mitochondrial HK enzymatic activity, lactate release and mitochondrial pyruvate levels. (unpaired T test, compared to Control condition) (O) Effect of 2-DG treatment on lactate release and mitochondrial levels of pyruvate in astrocyte. (unpaired T test) (P) Lactate release by Bsg knockdown or control astrocytes. (unpaired T test) (Q) EAE development in C57BL/6 or NOD mice that received lentiviral constructs to knockdown Bsg in astrocytes at the time points indicated by the arrows. (n≥7, N = 2, Regression slope T test). (R) Axonal loss and demyelination in spinal cords from NOD EAE mice. (unpaired T test)

Article Snippet: Mutagenesis and Cloning To generate expression plasmids for mutant or truncated overexpression plasmids, human PLA2G4A-His tag overexpression plasmid (#MG51714-CH, Sino Biological) and human MAVS-flag overexpression plasmid (#45905, Addgene) were used as templates.

Techniques: Inhibition, Pull Down Assay, Western Blot, Activity Assay, Binding Assay, Over Expression, Construct

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Miglustat levels in the CNS after oral administration of 600 mg/kg Miglustat. (unpaired T test) (B) EAE development in NOD mice treated with Miglustat (600 mg/kg administered orally before initiation of progressive phase, n≥6, N=3, Regression slope T test). (C-G) CNS samples were harvested 41 days after EAE induction from mice treated with Miglustat or vehicle as shown in Figure 6b. The number of CNS-infiltrating inflammatory monocytes (C, unpaired T test). Whole genome expression in astrocytes isolated from Miglustat treated NOD EAE mice (D; n = 6). Axonal loss and demyelination in spinal cord (E, unpaired T test). Immunoflourescence analysis of C3+GFAP+ astrocytes. Bar plots depict the number of C3+GFAP+ astrocyte within the observation field (F, unpaired T test, compared to EAE condition). Immunofluorescence analysis of NF-κB activation among MAVS+cPLA2+GFAP+ astrocytes (G, unpaired T test, compared to EAE condition). Bar plots depict the number of MAVS+cPLA2+acetyl-p65+GFAP+ astrocyte within the observation field. (H) mRNA expression determined by qPCR in activated human and mouse astrocytes in the presence of Miglustat. Data are shown relative to resting astrocytes. (unpaired T test) (I) Human and mouse astrocyte conditioned medium were analyzed using in vitro monocyte migration and neurotoxicity assays. Migrating monocytes and neuronal death in the resting vehicle-treated group were set as 100%. (unpaired T test) (J) mRNA expression in polarized microglia was determined by qPCR. Microglia was co-cultured with activated astrocytes. (unpaired T test) (K) Lactate release from activated astrocytes in the presence of Miglustat. (unpaired T test, compared to activated vehicle condition) (L) Effect of Miglustat treatment on cPLA2 enzymatic activity in astrocytes, evaluated by cPLA2 activity assay. (unpaired T test, compared to all)

Article Snippet: Mutagenesis and Cloning To generate expression plasmids for mutant or truncated overexpression plasmids, human PLA2G4A-His tag overexpression plasmid (#MG51714-CH, Sino Biological) and human MAVS-flag overexpression plasmid (#45905, Addgene) were used as templates.

Techniques: Expressing, Isolation, Immunofluorescence, Activation Assay, In Vitro, Migration, Cell Culture, Activity Assay

Journal: Cell

Article Title: Metabolic control of astrocyte pathogenic activity via cPLA2-MAVS

doi: 10.1016/j.cell.2019.11.016

Figure Lengend Snippet: (A) Immunostaining analysis of the co-localization (white arrowheads) of cPLA2 and mitochondria marker Tom20 in GFAP+ astrocytes in MS tissue. GFAP+ astrocytes in lesion, NAWM near lesion (20–50 μm from lesion) and NAWM far from lesion (>500 μm) (B) Immunostaining analysis of the co-localization (white arrowheads) of cPLA2 and MAVS in MS or HC tissue GFAP+ astrocytes. (C) Quantification of cPLA2+MAVS+ co-localization signal in GFAP+ and GFAP− fields. (n = 30–54 cells per condition, unpaired T test, compared to all) HC = healthy control, WM = white matter, GM = gray matter, NAWM = normally appearing white matter, NAGM = normally appearing gray matter.

Article Snippet: Mutagenesis and Cloning To generate expression plasmids for mutant or truncated overexpression plasmids, human PLA2G4A-His tag overexpression plasmid (#MG51714-CH, Sino Biological) and human MAVS-flag overexpression plasmid (#45905, Addgene) were used as templates.

Techniques: Immunostaining, Marker