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:

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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Anti-inflammatory Role of Carotenoids in Endothelial Cells Derived from Umbilical Cord of Women Affected by Gestational Diabetes Mellitus

doi: 10.1155/2019/8184656

Figure Lengend Snippet: Effect of carotenoids on TNF- α -induced monocyte interaction in C- and GD-HUVECs. Monocyte-HUVEC adhesion in C- and GD-HUVECs untreated (Basal) and incubated for 24 h with BC or Lyc (2.5 μ mol/L) and then stimulated for 16 h with or without TNF- α (1 ng/mL). In the histogram (upper side), quantitative data express the number of U937 cells adhering within a high-power field (3.5mm 2 ). Each measurement is expressed as the mean ± SD of adhering cells from 3 experiments ( n = 3), each consisting of 8 counts per condition. In the lower side, representative photos of C- and GD-HUVECs for each experimental condition. ANOVA and Bonferroni multiple comparison test: ∗ p < 0.05 vs. basal C-HUVECs, ∗∗ p < 0.05 vs. TNF- α C-HUVECs, ∗∗∗ p < 0.05 vs. Basal GD-HUVECs, # p < 0.05 vs. TNF- α GD-HUVECs. Student'st-test: † p < 0.0002 basal GD-HUVECs vs. basal C-HUVECs, ‡ p < 0.0001 TNF- α GD-HUVECs vs. TNF- α C-HUVECs.

Article Snippet: The cells were grown to confluence in six-well tissue culture plates and U937 cell lines (European Collection of Authenticated Cell Cultures (ECACC)) were used to evaluate the adhesion to HUVEC monolayers as previously described [ ].

Techniques: Incubation, Comparison