Journal: Molecular Medicine

Article Title: The vasoconstrictor adenosine 5′-tetraphosphate is a danger signal that induces IL-1β

doi: 10.1186/s10020-025-01116-6

Figure Lengend Snippet: Ap4 induces IL-1β release in immune cells. A and B PBMCs or C THP-1 monocytes were primed without or with Pam 3 CSK 4 (1 µg/ml) for 3 h and then stimulated with Ap4 (5 mM) for A and B 3 or C 6 h. Cell culture supernatants were analyzed ( A and C ) for IL-1β concentration by ELISA and B for LDH release. B Results are expressed as % of maximum LDH release. Mean ± SEM ( n = 4–5). One-way ANOVA followed by Dunnett´s post-test, ns ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. D – F THP-1 macrophages were primed with Pam 3 CSK 4 (1 µg/ml) for 3 h and then stimulated for 3 h with D increasing concentrations of Ap4 or E and F 5 mM Ap4. D IL-1β release in cell culture supernatants was determined by ELISA. Mean ± SEM ( n = 3–6). One-way ANOVA followed by Tukey´s post-test, **** P ≤ 0.0001. E Cell culture supernatants of stimulated THP-1 macrophages were transferred to HEK-blue IL-1β reporter cells. SEAP production was detected using QUANTI-Blue and optical density was measured at 620 nm. Mean ± SEM ( n = 4). One-way ANOVA followed by Dunnett´s post-test, ns ≥ 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. F Cell culture supernatants were analyzed for LDH release. Results are expressed as % of maximum LDH release. Mean + SEM ( n = 3). G , H THP-1 macrophages were stimulated G 24 h or H 4 h with Ap4 (5 mM) or Pam 3 CSK 4 (1 µg/ml). G IL-8 and H TNF release in cell culture supernatants was determined by ELISA. Mean ± SEM ( n = 3). I U937 macrophages were primed with Pam 3 CSK 4 (1 µg/ml) for 3 h and then stimulated with Ap4 (2 mM) or ATP (2 and 5 mM) for 3 h. IL-1β in cell culture supernatants was determined by ELISA. Mean ± SEM ( n = 3). One-way ANOVA followed by Tukey´s post-test, ns ≥ 0.05, **** P ≤ 0.0001

Article Snippet: THP-1 cells (ACC 16, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany), U937 cells (300368, Cell Lines Service GmbH, Eppelheim, Germany) or NLRP3-KO THP-1 cells (thp-konlrp3z, Invivogen, Toulouse, France) were cultured in growth medium consisting of RPMI 1640 (11530586, Fisher scientific, Schwerte, Germany) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (P4333, Sigma-Aldrich, Taufkirchen, Germany), 2 mM l -glutamine (G7513, Sigma-Aldrich, Taufkirchen, Germany) and 10% heat-inactivated fetal bovine serum (FBS; S0615, Sigma-Aldrich, Taufkirchen, Germany) at a density of 2–8 × 10 5 cells/ml.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Molecular Medicine

Article Title: The vasoconstrictor adenosine 5′-tetraphosphate is a danger signal that induces IL-1β

doi: 10.1186/s10020-025-01116-6

Figure Lengend Snippet: Ap4-induced IL-1β release is potassium- and NLRP3-independent. THP-1 macrophages were primed with Pam 3 CSK 4 (1 µg/ml) for 3 h and then stimulated with Ap4 (5 mM) A in the presence of potassium chloride (75 mM) or B without potassium chloride for 3 h. B NLRP3 inhibitors MCC950 (10 µM) and Bay 11-7082 (20 µM) were added 1 h before stimulation. IL-1β release in cell culture supernatants was determined by ELISA. IL-1β release induced by Ap4 was set to 100%. All other values were calculated accordingly. Mean ± SEM ( n = 3). One-sample t -test against 100%, ns ≥ 0.05. C NLRP3-KO THP-1 macrophages were primed with Pam 3 CSK 4 (1 µg/ml) for 3 h and then stimulated with Ap4 (5 mM) for 3 h. IL-1β concentration in the supernatants was analyzed by ELISA. Mean ± SEM ( n = 3). One-way ANOVA followed by Tukey´s post-test, **** P ≤ 0.0001. D U937 macrophages were primed with Pam 3 CSK 4 (1 µg/ml) for 3 h and then stimulated with Ap4 (2 mM) or ATP (5 mM) for 3 h. The NLRP3 inhibitor MCC950 (40 µM) was added 1 h before stimulation. IL-1β release in cell culture supernatants was determined by ELISA. Ap4-induced IL-1β release was set to 100%. All other values were calculated accordingly. Mean ± SEM ( n = 3–4). One-sample t -test against 100%, ns ≥ 0.05

Article Snippet: THP-1 cells (ACC 16, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany), U937 cells (300368, Cell Lines Service GmbH, Eppelheim, Germany) or NLRP3-KO THP-1 cells (thp-konlrp3z, Invivogen, Toulouse, France) were cultured in growth medium consisting of RPMI 1640 (11530586, Fisher scientific, Schwerte, Germany) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (P4333, Sigma-Aldrich, Taufkirchen, Germany), 2 mM l -glutamine (G7513, Sigma-Aldrich, Taufkirchen, Germany) and 10% heat-inactivated fetal bovine serum (FBS; S0615, Sigma-Aldrich, Taufkirchen, Germany) at a density of 2–8 × 10 5 cells/ml.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

doi: 10.3389/fimmu.2018.03153

Figure Lengend Snippet: Flow cytometry analysis of target binding specificity by 8F4 bi-specific antibody. Each data point represents the mean of triplicate measures and error bars represent SEM. (A) Flow cytometry analysis of 8F4 bi-specific antibody binding of CD3 + Jurkat and CD3 − (J.RT3) cell lines and (B) CD5 + and CD5 − normal healthy donor peripheral blood lymphocytes in a dose dependent manner. Mean fluorescent intensity is reported. (C–F) Flow cytometry analysis of 8F4 bi-specific antibody binding of different PR1/HLA-A2 + (T2 PR1, THP1, U937 A2 + , K562 A2 + ) and control (T2 CMV, U937 WT, K652 WT) cell lines, detected with anti-HisTag PE. Mean fluorescent intensity is reported. These results indicate target-specific PR1/HLA-A2 and human CD3 cell surface binding. Data combined from 2 independent experiments in triplicate or quadruplicate were shown.

Article Snippet: Cell lines OKT3, Chinese hamster ovary (CHO), T2 (TAP-deficient), Jurkat, J.RT3-T3.5 (Jurkat cell line lacking functional CD3), THP1, U937, and K562 were obtained from ATCC (Manassas, VA, United States).

Techniques: Flow Cytometry, Binding Assay, Control

Journal: Frontiers in Immunology

Article Title: A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

doi: 10.3389/fimmu.2018.03153

Figure Lengend Snippet: T-cell activation and cytokine production by 8F4 bi-specific antibody. Eighteen hour co-culture experiments combining 8F4 bi-specific antibody with healthy donor lymphocyte effectors and target AML cell lines U937 A2+, U937 WT, and THP1 at an E:T ratio of 2:1 were conducted in triplicate. Bi-specific antibodies led to activation of human PBL in the presence of PR1/HLA-A2 in a dose dependent manner, and activated human PBL produced various inflammatory cytokines only in the presence of 8F4 bi-specific antibody and target AML cells. Flow cytometry assessed surface CD69 on activated T-cells in the presence of target cells as compared to control experiments lacking U937 (A) and THP1 (B) AML targets. Cytokines from co-culture experiments were then quantified by ELISA for TNFα (C) , IFNγ (D) , and IL-6 (E) . Each data point represents the mean of triplicate measures and error bars represent SEM. One representative data of 2 independent experiments were shown.

Article Snippet: Cell lines OKT3, Chinese hamster ovary (CHO), T2 (TAP-deficient), Jurkat, J.RT3-T3.5 (Jurkat cell line lacking functional CD3), THP1, U937, and K562 were obtained from ATCC (Manassas, VA, United States).

Techniques: Activation Assay, Co-Culture Assay, Produced, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: A Novel T-Cell Engaging Bi-specific Antibody Targeting the Leukemia Antigen PR1/HLA-A2

doi: 10.3389/fimmu.2018.03153

Figure Lengend Snippet: Redirected cytotoxicity of T-cells against PR1/HLA-A2 + cell lines. Target AML cells were pre-stained with pacific blue dye and following co-incubation with 8F4 bi-specific antibody and healthy donor PBL at an E:T ratio of 2:1, cells were stained with a fixable live/dead stain. Cytotoxicity calculations were based on total live pacific blue positive cells counts. Calculated % cytotoxicity for co-culture with AML U937 A2 + or U937 WT (A) and THP1 (B) cell lines is presented, based on flow cytometry analysis. These data indicate the 8F4 bi-specific antibody initiated dose-dependent AML-specific cytotoxicity after only 18 h co-culture. Each data point represents the mean of triplicate measures and error bars represent SEM. One representative data of 2 independent experiments were shown.

Article Snippet: Cell lines OKT3, Chinese hamster ovary (CHO), T2 (TAP-deficient), Jurkat, J.RT3-T3.5 (Jurkat cell line lacking functional CD3), THP1, U937, and K562 were obtained from ATCC (Manassas, VA, United States).

Techniques: Staining, Incubation, Co-Culture Assay, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 1. UNBS1450 induces apoptotic cell death in U937 cells. (A) Molecular structure. (B) Analysis of UNBS1450-induced cell death was performed by Trypan Blue staining after 24, 48 and 72 h of treatment with UNBS1450 at 10, 15, 20 and 30 nM. (C) Cell cycle analysis after 24 h of incubation time with indicated concentrations. (D) Hoechst staining (upper panel) and quantification (lower panel) of the fraction of cells presenting fragmented nuclei. (E) Flow cytometry analysis after 24 h of incubation time at indicated concentrations. (F) Analysis of PBMCs viability after UNBS1450 treatment. PBMCs were seeded at 2 106, then after 24 h treated for 24 h with various concentrations (0–100 nM) of UNBS1450. Cells were then stained either by Trypan Blue (upper panel) or by Hoechst (panel below) to analyze either cell integrity or apoptosis induction. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Staining, Cell Cycle Assay, Incubation, Flow Cytometry

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 2. Na+/K+-ATPase subunit a1 mRNA quantification. Na+/K+-ATPase subunit a1 mRNA content of untreated PBMCs and a wide panel of hematological cancer cell lines including K562, Jurkat and U937 cells was transcribed and then quantified by RT-PCR. The quantification of three independent experiments is expressed in brute 2^DCt values S.D.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 3. (A) Caspase activation. U937 cells were incubated in RPMI + 10% FCS UNBS1450 20 nM up to 24 h. Western blot analysis of UNBS1450-induced cleavage of pro-caspases-9, -8, -7 and -3. (B) Analysis of expression levels of anti- apoptotic proteins. UNBS1450-induced expression level alterations of XIAP, Bcl-2 and Mcl-1. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Activation Assay, Incubation, Western Blot, Expressing

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 4. UNBS1450 enables Bak/Bax activation. U937 cells were incubated for 24 h in RPMI + 10% FCS in presence or in absence of UNBS1450. Bak (A.) and Bax (B.) activation status were assessed by using primary antibodies specifically targeting activated forms of Bak (Ab-1; Calbiochem) and Bax (6A7; Santa Cruz). Counterstaining was done by Hoechst staining to assess apoptotic nuclei. The data shown here were representative for three independent experiments with similar results.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Activation Assay, Incubation, Staining

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 5. Inhibition by UNBS1450 of TNFa-induced NF-kB activation. (A) K562 and (B) Jurkat cells were pretreated with UNBS1450 at various concentrations from 10 to 50 nM and incubated for 2 h, followed by TNFa addition (20 ng/ml) and an additional incubation period of 6 h. Results are represented as the ratio of the measured luminescence of the firefly luciferase vector divided by the measured luminescence of the Renilla plasmid. Untreated cells were used as a negative control, cells treated with TNFa only as a positive control. Results are presented as mean S.D. of 3 individual measurements performed in triplicates. (C) Effect of UNBS1450 on the binding affinity of NF-kB was assessed by an EMSA on the K562 and Jurkat cell lines. The data shown here were representative for three independent experiments with similar results. (D) For supershift/immunodepletion experiments, the nuclear extracts and labelled probes were incubated in the reaction mixture for 30 min on ice prior to a 30 min incubation with 2 mg of anti-p50 or anti-p65 antibodies. SS designates supershifted bands. (E) Jurkat cells were incubated with UNBS1450 (40 nM) for 2 h, followed by a TNFa (20 ng/ml) activation for the indicated time periods. Cytoplasmic and nuclear extracts were prepared, fractionated on a 10% SDS-page gel, transferred to a membrane and then tested for IkBa and p65. Protein loading and purity of nuclear/cytosolic extracts were verified by lamin B and a-tubulin Western blots. Data shown are representative for three independent experiments with similar results. K562 (F), and U937 (G) cells were incubated for 2 h in RPMI + 10% FCS in presence or in absence of various concentrations (10–50 nM) of UNBS1450 before being activated by TNFa during 22 h. After 24 h of incubation IL-8 concentrations in supernatants were measured. Untreated cells served as negative control whereas cells activated by TNFa only were used as a positive control. The data shown here were representative for three independent experiments with similar results.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Inhibition, Activation Assay, Incubation, Luciferase, Plasmid Preparation, Negative Control, Positive Control, Binding Assay, Immunodepletion, SDS Page, Membrane, Western Blot

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: VEGF-A mRNA interacts with the GAIT complex. (A) Secondary structure and sequence features of the human Cp GAIT element (top panel). The query pattern, based on the secondary structure and sequence features of the Cp GAIT element, was used to search a nonredundant 3′UTR database using the PatSearch program (bottom panel). Following the syntax of the PatSearch algorithm, allowed base-pairs are represented by rnumber and patterns defined by pnumber. The GAIT element-specific stems and loops are shown below. (B) PatSearch result predicted the presence of GAIT elements in Cp and VEGF-A 3′UTR. UTRdb ID refers to the sequence entry in the UTR database, and sequence position refers to the 3′UTR position of the sequence encoding the predicted GAIT element. (C) To show VEGF-A mRNA interaction with the GAIT complex in vivo, U937 cells were treated with IFN-γ for 8 or 24 h, and lysates were immunoprecipitated (IP) with anti-EPRS antibody to isolate GAIT complex, or with control pre-immune (Pre-im.) serum. RNA associated with the GAIT complex, or present in the non-immunoprecipitated supernatant (Sup.), was subjected to RT–PCR using primers specific for VEGF-A or β-actin mRNA, and products were resolved in 1.6% agarose gels. (D) To verify antibody specificity, lysate from U937 cells treated with IFN-γ for 24 h was immunoprecipitated with polyclonal anti-human EPRS antibody and immunoblotted with the same antibody, or with pre-immune serum as control.

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: Sequencing, In Vivo, Immunoprecipitation, Control, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: Translational silencing of VEGF-A expression in vivo. (A) RT–PCR analysis of total RNA from U937 cells treated with IFN-γ for 0, 8, or 24 h. RT–PCR was done using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). Real-time PCR results indicating the increase in VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells are included below the top panel (expressed as fold-increase normalized to β-actin). (B) Cell lysates from U937 cells treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. (C) RT–PCR analysis of total RNA from human PBMCs treated with IFN-γ for 0, 8, or 24 h. RT–PCR was performed using primers specific for VEGF-A (top panel) and GAPDH (bottom panel). (D) Cell lysates from PBMCs treated with IFN-γ for 0, 8, or 24 h were processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top) and anti-GAPDH (bottom panel) antibodies. (E) U937 cells were treated with IFN-γ for up to 24 h. At the end of each interval, cells were metabolically labeled with [35S]Met/Cys for 1 h. Conditioned media and cell lysates were immunoprecipitated with anti-VEGF-A antibody and resolved by electrophoresis on SDS–10% polyacrylamide gel (top panel). Monomeric and dimeric VEGF-A forms are indicated by arrows. The same samples were subjected to electrophoresis without immunoprecipitation (bottom). (F) U937 cells were treated with IFN-γ for 8 or 24 h and cytosolic extracts were fractionated into polysomal and non-polysomal, RNP fractions by ultracentrifugation on a 20% sucrose cushion in the presence or absence of 10 mM EDTA. RNA associated with each fraction was isolated and subjected to RT–PCR using primers specific for VEGF-A (top panel) and GAPDH (bottom panel).

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: Expressing, In Vivo, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Metabolic Labelling, Labeling, Immunoprecipitation, Electrophoresis, Isolation

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: The 3′UTR of VEGF-A mRNA mediates translation inhibition. (A) Schematic of VEGF-A mRNA and chimeric luciferase constructs used for in vitro translation (top panel). The m7G cap is indicated by an open circle, the IRES by a light gray rectangle, the putative GAIT element by a black rectangle, and the AREs by dark gray rectangles. Capped, FLuc-VEGF-A 3′UTR(11–900)-A30 RNA was translated in RRL containing [35S]Met, and in absence or presence of cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h (middle panel). Capped, RLuc RNA lacking the GAIT element was co-translated in each reaction as control. Translation reactions were resolved on SDS–10% polyacrylamide gel. The same RNAs were translated in the presence of cytosolic extract from 24-h, IFN-γ-treated U937 cells, and in the presence of 10- and 50-fold molar excess of in vitro transcribed VEGF-A 3′UTR RNA as competitor (bottom panel). (B) Schematic of chimeric luciferase constructs used for in vitro translation (top panel). In vitro translation, in presence of IFN-γ-treated U937 cytosolic extracts, of capped FLuc-VEGF-A 3′UTR(324–455)-A30 encompassing the putative GAIT element (middle panel), and FLuc-VEGF-A 3′UTR(441–560)-A30 (bottom panel). RLuc RNA was co-translated in each reaction.

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: Inhibition, Luciferase, Construct, In Vitro, Control

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: Functional identification of the VEGF-A 3′UTR GAIT element. (A) Folding structures of the Cp (nt 78–106) and the putative VEGF-A GAIT (nt 358–386) elements as predicted by the Mfold algorithm. Base pairing between A7:U23 and U8:A22 was disallowed while folding the VEGF-A GAIT element. (B) Chimeric luciferase constructs containing wild-type or mutant VEGF-A 3′UTR GAIT elements. Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element (FLuc-VEGF-A GAIT-A30) or a mutant (U10C) GAIT element (FLuc-VEGF-A GAITmut-A30), downstream of FLuc (top panel), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated U937 cells (bottom panel). RLuc RNA was co-translated in each reaction. (C) Capped and poly-A tailed RNAs, containing the putative VEGF-A GAIT element or a mutant GAIT element as in (B), were subjected to in vitro translation in presence of cytosolic extracts from IFN-γ-treated human PBMC (top panel). RLuc RNA was co-translated in each reaction. Fluc was quantified by densitometry, normalized to Rluc, and expressed as per cent of control condition without cell lysate (bottom). (D) U937 cells were transfected with eukaryotic, CMV-driven expression vectors containing the FLuc gene upstream of either wild-type (CMV-FLuc-VEGF-A GAIT-A30) or mutant VEGF-A GAIT element (CMV-FLuc-VEGF-A GAITmut-A30) or lacking any GAIT element (CMV-FLuc). Cells were co-transfected with a vector containing RLuc gene under the SV40 promoter. Following transfection, cells were treated with IFN-γ for 8 (gray bars) or 24 h (black bars), or with medium alone (hatched bars). Luciferase activity in cell lysates was measured by dual luciferase assay. Results show mean and standard deviation of values from three independent experiments.

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: Functional Assay, Luciferase, Construct, Mutagenesis, In Vitro, Control, Transfection, Expressing, Plasmid Preparation, Activity Assay, Standard Deviation

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: The GAIT complex binds the VEGF-A GAIT element and causes translational silencing. (A) RNA EMSA using 32P-labeled Cp and VEGF-A GAIT element probes. The riboprobes were incubated with cytosolic extracts from U937 cells treated with IFN-γ for up to 24 h. RNA–protein complexes were resolved by electrophoresis on a nondenaturing 5% polyacrylamide gel. (B) RNA–protein complexes formed between 32P-labeled VEGF-A GAIT element RNA and lysates from 24-h, IFN-γ-treated U937 cells were supershifted with antibodies against GAIT complex components. The cell lysate was incubated with the respective antibodies or non-immune IgG before incubation with the riboprobe. (C) Lysate from U937 cells treated with IFN-γ for 24 h was incubated with protein-A Sepharose beads coupled to anti-EPRS antibody (or to pre-immune serum, Pre-im.) to immunodeplete the GAIT complex. The beads were pelleted, and the supernatant subjected to immunoblotting with anti-EPRS antibody to verify effective immunodepletion. (D) At 24-h, IFN-γ-treated U937 cell lysates, immunodepleted with anti-EPRS antibody or pre-immune serum, were added to in vitro translation reactions containing FLuc-VEGF-A 3′UTR(11–900)-A30 and RLuc RNAs.

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: Labeling, Incubation, Electrophoresis, Western Blot, Immunodepletion, In Vitro

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: Ablation of the GAIT complex in vivo prevents translational silencing of VEGF-A. (A) Lysates from U937 cells stably transfected with pSUPER vector (U937-pSUPER) or pSUPER encoding a short hairpin RNA targeting L13a (U937-L13a-SHR) were immunoblotted with anti-L13a antibody. (B) Lysates from the stably transfected cell lines in (A) were treated with IFN-γ for 0, 8, or 24 h and processed in Laemlli gel-loading buffer in absence of reducing agent. Lysates were subjected to immunoblotting with anti-VEGF-A (top panel) and anti-GAPDH (bottom panel) antibodies. (C) Total RNA was isolated from the stably transfected cell lines treated with IFN-γ for 0, 8, or 24 h, and analyzed by RT–PCR using primers specific for VEGF-A (top panel) and β-actin (bottom panel). Real-time PCR results indicating increased VEGF-A mRNA expression in IFN-γ-treated cells compared to untreated cells (expressed as fold-increase normalized to β-actin) are inserted below the top panel. (D) The cell lines described in (A) were treated with IFN-γ for 24 h and lysates immunoprecipitated with anti-EPRS antibody, followed by RT–PCR with VEGF-A-specific primers.

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: In Vivo, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Expressing, Immunoprecipitation

Journal:

Article Title: A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

doi: 10.1038/sj.emboj.7601774

Figure Lengend Snippet: Silencing of VEGF-A translation in monocytic cells inhibits angiogenic activity. (A) EC proliferation was measured in presence of medium conditioned by IFN-γ-treated U937 cells. U937 cells were pre-treated with IFN-γ for up to 24 h, and then fresh medium was added for an additional 2 h. The conditioned medium was added to 50% confluent ECs, and proliferation measured by MTT assay. Cells were treated with recombinant VEGF-A (rVEGF-A, 10 ng/ml) as a positive control. Stimulation of proliferation was expressed as fold-increase compared to cells treated with medium alone (gray bars). Parallel wells contained conditioned medium pre-incubated with anti-VEGF-A antibody (black bars). Shown are the mean and standard deviation from three independent experiments. (B) Tube-formation by ECs on growth factor-depleted matrigel was determined after 12 h in presence of conditioned medium from U937 cells treated with IFN-γ for 8, 16, or 24 h, or with recombinant human VEGF-A (10 ng/ml). (C) EC tube formation was quantitated by computer-assisted tracing. Shown are the mean and standard deviation from three representative fields, for three independent experiments. (D) IFN-γ activates the transcription of VEGF-A, Cp, and other pro-inflammatory genes in macrophages at the site of chronic inflammation. Subsequently, IFN-γ activates the GAIT complex that binds to the GAIT element in the 3′UTR of VEGF-A, Cp, and possibly other transcripts, and silences their translation. This mechanism prevents persistent expression of these inflammatory proteins and reduces or resolves chronic inflammation and tissue injury.

Article Snippet: [α- 32 P]UTP-labeled VEGF-A or Cp GAIT element RNA was incubated with cytosolic extracts from U937 cells incubated with IFN-γ for up to 24 h. For supershift assays, the lysate was pre-incubated with affinity-purified anti-EPRS, anti-L13a ( Mazumder et al , 2003 ), anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NSAP (Anaspec, San Jose, CA, USA) polyclonal antibodies, or with IgG purified from pre-immune rabbit serum.

Techniques: Activity Assay, MTT Assay, Recombinant, Positive Control, Incubation, Standard Deviation, Expressing