ep4 antagonist ono ae3 208  (Tocris)

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Overproduction of PGE 2 impairs MAS activity and mitochondrial dynamics (A) Gene expression of E-type prostanoid receptors in resting PMs. TPM, transcripts per kilobase of exon model per million mapped reads. N = 3 biological replicates. (B) Inhibition of EP4 with ONO-AE3-208, but not EP2 with PF-04418948, elevates the protein levels of MAS components Slc25a12 and Slc25a13 in Spns2 −/− PMs. N = 3 biological replicates. (C) EP4 activation contributes to the downregulation of Slc25a12 and Slc25a13 in WT PMs exposed to either PGE 2 or Spns2 inhibitor SLF1081851. N = 3 biological replicates. ( D , E ) Flow cytometry analysis of overlaid Δψm probed by MitoTracker™ Red (MT, D) and mitochondrial mass probed by CytoFix™ MitoRed (CF, E). MFI, mean fluorescent intensity. (F) Inhibition of EP4 restores the average Δψm (calculated by the ratio of MT/CF) in Spns2 −/− PMs. N = 3 biological replicates ( D to F) . (G) EP4 inhibition modulates the expression of mitochondrial dynamics-related proteins, promoting mitochondrial fusion in Spns2 −/− PMs. N = 3 biological replicates. (H) Transmission electron microscopy reveals that EP4 inhibition facilitates mitochondrial fusion in Spns2 −/− PMs. Scale bar = 1 μm. Arrowheads indicate fused (red) and fragmented (green) mitochondrial morphology. N = 3 biological replicates. Data are presented as mean ± s.e.m. P values were determined by unpaired t -test (A) and one-way ANOVA with Sidak’s correction for multiple comparisons ( B to H) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 µM S1P (Cayman Chemical, 62570), 1 µM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 µM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activity Assay, Expressing, Inhibition, Activation Assay, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Excessive EP4 activation impairs mitochondrial respiration and increases oxidative stress in Spns2 −/− PMs (A) Blocking EP4 with ONO-AE3-208 increases the oxygen consumption rates (OCR) in Spns2 −/− PMs. (B) Quantitative analysis of basal respiration, maximal respiration, ATP production, and proton leakage reveal the restoration of mitochondrial respiration following EP4 blockade. N = 4 biological replicates ( A and B) . (C) EP4 inhibition reduces intracellular lactate levels in Spns2 −/− PMs. N = 12 biological replicates. (D) Flow cytometry analysis reveals a decrease in MitoSOX™ Red-probed mtROS generation in ONO-AE3-208-treated Spns2 −/− PMs. N = 3 biological replicates. (E) Total intracellular ROS probed by CellROX ® Orange remains comparable among each group. N = 3 biological replicates. (F) EP4 inhibition diminishes the activities of total superoxide dismutase (SOD) and catalase, indicating alleviated oxidative stress in Spns2 −/− PMs. N = 6 biological replicates. Data in the panels A , B , D , and E are presented as mean ± s.e.m. In panels C and F , the central bands represent the median values, the boxes represent the distance between the third and the first quartile, and the whiskers represent the ranges between the minimum and maximum values. P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons. * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 µM S1P (Cayman Chemical, 62570), 1 µM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 µM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Activation Assay, Blocking Assay, Inhibition, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: PGE 2 contributes to the early-phase hyperinflammation during bacterial infections ( A , B ) Flow cytometry analysis shows reduced mtROS generation probed by MitoSOX™ Red (A ) and decreased total intracellular ROS probed by CellROX ® Orange (B) in ONO-AE3-208-treated Spns2 −/− PMs at 3-h post-LPS challenge. N = 3 biological replicates ( A and B ). (C) EP4 blockade reduces the gene expression of inflammatory cytokines within 3-h post-LPS challenge due to the suppression of the lactate-ROS axis. Notably, EP2 blockade also attenuates the early-phase hyperinflammation, possibly via a mechanism independent of the lactate-ROS axis. Both EP2 and EP4 blockade partially restore the suppressed gene expression of inflammatory cytokines in Spns2 −/− PMs after 6-h post-LPS challenge. N = 3 biological replicates. (D) Schematic of the in vivo experiments using heat-killed E. coli -induced peritoneal infection models. (E, F) Both EP2 and EP4 inhibition alleviate hyperinflammation (E) and significantly improve survival rates (F) in Spns2 −/− sepsis models triggered by intraperitoneal infection with heat-killed E. coli . N = 6 biological replicates for cytokine measurement. N = 6 to 8 biological replicates for survival analysis. Data are presented as mean ± s.e.m. ( A , B , and E ) and percentage (F) . P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons ( A , B , and E ) and log-rank test adjusted by the Bonferroni method (F) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant. # indicates P value is less than the Bonferroni-corrected threshold

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 µM S1P (Cayman Chemical, 62570), 1 µM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 µM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Flow Cytometry, Expressing, In Vivo, Infection, Inhibition

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Excessive PGE 2 production induces immunosuppression as infection progresses (A) Spns2 −/− PMs exhibit significantly elevated gene expression of Ptges compared to WT PMs before and after the LPS challenge. TPM, transcripts per kilobase of exon model per million mapped reads. N = 3 biological replicates. (B) Spns2 −/− PMs release higher levels of PGE 2 than WT PMs within 6-h post-LPS challenge. N = 6 biological replicates. (C) Gene expression of E-type prostanoid receptors in PMs at 3-h post-LPS challenge. N = 3 biological replicates. (D) Blockade of both EP2 and EP4 enhances TNFα and IL-6 release by Spns2 −/− PMs within 12-h post-LPS challenge. N = 4 biological replicates. (E) Schematic of the in vivo experiments using CLP models. (F) Survival curves from CLP models demonstrate that partial recovery of the inflammatory response induced by either EP2 or EP4 blockade improves the survival of Spns2 −/− rats. N = 7 to 12 biological replicates. (G) The levels of serum pro-inflammatory cytokines measured at 36-h post-infection indicate that EP2 or EP4 inhibition is effective but insufficient to overcome immunosuppression in Spns2 −/− CLP models. N = 4 biological replicates. (H) Colony-forming units (CFU) counts in livers and spleens at 36-h post-infection reveal higher bacterial loads in EP2- and EP4-inhibited Spns2 −/− CLP models. N = 5 to 6 biological replicates. Data are presented as mean ± s.e.m. ( A to D , G , and H ) and percentage (F) . P values were determined by unpaired t -test ( A to C ), one-way ANOVA with Sidak’s correction for multiple comparisons ( D , G , and H ), and log-rank test adjusted by the Bonferroni method (F) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant. # indicates P value is less than the Bonferroni-corrected threshold

Article Snippet: Spns2 −/− models were treated with 1 ml of 10 µM S1P (Cayman Chemical, 62570), 1 µM EP2 antagonist PF-04418948 (TargetMOI, T3306) [ ], or 1 µM EP4 antagonist ONO-AE3-208 (TargetMOI, TQ0290) [ ] immediately after infection.

Techniques: Infection, Expressing, In Vivo, Inhibition

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Heatmap showing relative expression of COX2 and type I ISGs in mice treated with either vehicle (Veh) or doxorubicin (Doxo) (GEO: GSE223698). (B) Heatmap showing relative expression of COX2 and type I ISGs in proliferating cells (Prof) and senescence cells (Sen) (GEO: GSE196610). (C) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for either 24 or 48 hours. Cell lysates were subjected to RT-qPCR to assess mRNA levels of COX2 (n = 3). (D) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1) for either 24 or 48 hours in the presence or absence of 1 µM celecoxib (COX2i). Cell lysates were collected and analyzed by ELISA to measure extracellular PGE 2 levels (n = 3). (E) A schematic illustrates PGE 2 -cAMP-PKA signaling. (F) THP-1 macrophages were treated with 1 µM PGE 2 in the presence or absence of 1 µM EP4 inhibitor (EP4i) for 16 hours. Cell lysates were collected and analyzed by ELISA to measure intracellular cAMP levels (n = 3). (G) THP-1 macrophages were treated with 1 µM PGE 2 at indicated concentrations in the presence or absence of 1 µM EP4 inhibitor (EP4i) for 16 hours. Cell lysates were collected and subjected to immunoblotting with the indicated antibodies. (H) THP-1 macrophages expressing ISRE-Luciferase were mock-infected or infected with HSV-1 (MOI = 1) in the presence of either 1 µM PGE 2 or 1 µM forskolin and 50 µM IBMX. At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (I-J) THP-1 macrophages were mock-infected or infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 24 h.p.i, cell lysates were subjected to RT-qPCR to assess mRNA levels of IFNβ (n = 3) (I) , and cell supernatants were subjected to ELISA to measure secreted levels of IFNβ (n = 3) (J) . (K) THP-1 macrophages expressing ISRE-Luciferase were mock-infected or infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 16 h.p.i, cell lysates were collected and subjected to a luciferase reporter assay to assess ISRE promoter activity (n = 3). (L) THP-1 macrophages were infected with HSV-1 (MOI = 1), followed by 1 µM PGE 2 stimulation in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi). At 16 h.p.i, cell lysates were collected and subjected to RT-qPCR to assess HSV-1 UL30 genomic abundance (n = 3). Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. P -values are indicated.

Article Snippet: For inhibition studies, cells were pre-incubated with EP4 antagonist ONO AE3-208 (Tocris 3565), PKA inhibitor BLU0588 (TargetMol, T60169), during the starvation period.

Techniques: Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: bioRxiv

Article Title: The COX2-PGE2-PKA Axis Suppresses Antiviral Immunity by Inhibiting mtDNA-Dependent STING Activation

doi: 10.64898/2026.04.03.716411

Figure Lengend Snippet: (A) Proteomic workflow in HEK293 cells with doxycycline-inducible expression of PKA Cα wild-type (WT) or W197R mutant (MUT). (B) The top 8 significantly enriched KEGG pathways within the mitochondrial quality control category identified from the proteomic dataset. (C) Schematic of the mt-mKeima mitophagy reporter. (D) Representative Airyscan live-cell imaging of THP-1 macrophages expressing mt-mKeima sensor treated with 1 µM PGE 2 in the presence of either 1 µM EP4 inhibitor (EP4i) or 1 µM PKA inhibitor (PKAi) for 16 hours. Scale bar, 10 µm. (E) Quantification of mitolysosome numbers shown in (D) (n = 10). Data were quantified from one representative experiment of three. (F) Representative images of live-cell 4D lattice light sheet imaging on THP-1 macrophages treated as in (D) . Scale bar, 20 µm. (G-H) Quantification of net mitolysosome displacement (n = 10) (G) and average mitolysosome speed (n = 12) (H) from imaging in (F) . (I) THP-1 macrophages treated with PGE 2 at indicated concentrations in the presence or absence of 1 µM PKA inhibitor (PKAi) for 16 hours. Mitochondrial fractions were isolated and subjected to immunoblotting with indicated antibodies. Band intensities were quantified and normalized to COX4 expression for PINK1 (J) and pUB Ser65 (K) (n = 3). (L) Working model illustrating that PGE 2 induces mitophagy and mitochondrial biogenesis to enhance mitochondrial homeostasis in a EP4- and PKA-dependent manner. Data are presented as mean ± s.e.m. Statistical significance was determined by one-way ANOVA followed by Sidak’s multiple comparisons test. p -values are indicated.

Article Snippet: For inhibition studies, cells were pre-incubated with EP4 antagonist ONO AE3-208 (Tocris 3565), PKA inhibitor BLU0588 (TargetMol, T60169), during the starvation period.

Techniques: Expressing, Mutagenesis, Control, Live Cell Imaging, Imaging, Isolation, Western Blot

Journal: Biomedicines

Article Title: TRAP-Induced Platelet Reactivity Is Inhibited by Omega-3 Fatty Acid-Derived Prostaglandin E3 (PGE3).

doi: 10.3390/biomedicines12122855

Figure Lengend Snippet: Figure 1. Effects of selective prostanoid receptor antagonists on the PGE3-mediated changes in TRAP-6-induced platelet aggregation. The effects of PGE3 in the absence and presence of the EP3 antagonist DG-041 (3 µM), and the EP4 antagonist ONO-AE3-208 (1 µM) on platelet aggregation induced by TRAP-6 (10 µM), over a 4 min period, in whole blood. Aggregation was determined by flow cytometry and measured by single platelet counting method. The results shown are the mean ± SEM of 6 experiments. *** p = 0.0004, **** p < 0.0001.

Article Snippet: The EP4 receptor antagonist ONO-AE3-208 and the EP3 receptor antagonist DG-041 were obtained from Biotechne® Tocris (Bristol, UK).

Techniques: Flow Cytometry

Journal: Biomedicines

Article Title: TRAP-Induced Platelet Reactivity Is Inhibited by Omega-3 Fatty Acid-Derived Prostaglandin E3 (PGE3).

doi: 10.3390/biomedicines12122855

Figure Lengend Snippet: Figure 2. Effects of selective prostanoid receptor antagonists on PGE3-mediated changes in TRAP-6- induced P-selectin expression. The effects of PGE3 in the absence and presence of the EP3 antagonist DG-041 (3 µM), and the EP4 antagonist ONO-AE3-208 (1 µM), on platelet P-selectin expression induced by TRAP-6 (10 µM), over a 4 minute period, in whole blood. P-selectin was measured by flow cytometry and is presented as median fluorescence (mf). The results shown are the mean ± SEM of 6 experiments. * p < 0.05, ** p < 0.005.

Article Snippet: The EP4 receptor antagonist ONO-AE3-208 and the EP3 receptor antagonist DG-041 were obtained from Biotechne® Tocris (Bristol, UK).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: Biomedicines

Article Title: TRAP-Induced Platelet Reactivity Is Inhibited by Omega-3 Fatty Acid-Derived Prostaglandin E3 (PGE3).

doi: 10.3390/biomedicines12122855

Figure Lengend Snippet: Figure 3. Effects of selective prostanoid receptor antagonists on the PGE3 mediated changes in TRAP-6-induced VASP-phosphorylation. The effects of PGE3 in the absence and presence of the EP3 antagonist DG-041 (3 µM), and the EP4 antagonist ONO-AE3-208 (1 µM) on VASP phosphorylation, over a 4 min period, in whole blood. VASP-phosphorylation was determined by flow cytometry using a cytometric bead array (VASPFix) and is presented as median fluorescence (mf). The results shown are the mean ± SEM of 6 experiments. ** p < 0.005, *** p = 0.0008, **** p < 0.0001.

Article Snippet: The EP4 receptor antagonist ONO-AE3-208 and the EP3 receptor antagonist DG-041 were obtained from Biotechne® Tocris (Bristol, UK).

Techniques: Phospho-proteomics, Flow Cytometry, Fluorescence