16 16 dimethyl pge2  (R&D Systems)

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 2. Involvement of PGE2 in NEC in human intestinal tissue. (A) The typical features of assessed samples within the jejunum and ileum segments; (B) PGE2 levels were measured in ileal homogenates from human patients. PGE2 levels in intestinal tissue lysates calculated per µg of protein. *P < 0.01, #P < 0.01 vs. non-NEC control; (C) Representative morphology of the human ileum (the area of necrosis and perforation, less affected ileum, nonaffected ileum and no-NEC control) using hematoxylin and eosin staining. Scale bars: 100 µm; (D) Representative immunofluorescence staining for COX-2 in the terminal ileum of NEC patients and non-NEC control patients (white arrows). Scale bars: 100 µm. Three independent experiments were repeated with similar results. The mRNA expression levels of EP1 (E), EP2 (F), EP3 (G), and EP4 (H) relative to β-actin mRNA were assessed in intestinal tissues using qRT‒PCR. Values represent means ± SEMs. *P < 0.01; #P < 0.01 (one-way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: Control, Staining, Immunofluorescence, Expressing

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 3. PGE2 administration attenuated the severity of experimental NEC. (A) PGE2 administration reduced the incidence of experimental NEC (damage scores over 2) in pups under hypothermic and hypoxic conditions. Column values represent the average of multiple experiments (n = 10–12 mice per group). Bars, SEM *P < 0.01 (one-way ANOVA); (B) Weight change of established NEC treatments as indicated. Columns, the average values of multiple experiments (n = 10–12 mice per group); bars, SEM, *P < 0.01, vs. control; #P < 0.01 (one-way ANOVA); (C) Kaplan‒Meier analysis for the pups among the different managements. Data represent percent survival (n = 10–18 mice per group); (D) Quantification of blood bacterial growth from pups managed as indicated. Values represent means ± SEMs. *p < 0.01 vs. the corresponding WT control; #P < 0.01 vs. the corresponding WT mice with NEC (one-way ANOVA); (E) Representative terminal ileums with H&E staining for the pups from three independent replicate coverslips with experimental NEC treated as indicated. Scale bars: 100 μm; (F) The severity scores for NEC were measured by a pathologist (n = 10–18 mice per group) according to morphological performance (E). Columns, means; bars, SEM. *P < 0.01; #P < 0.01 (one-way ANOVA). The mRNA levels of IL-6 (G) and TNFα (H) in the intestinal tissues were measured from the pups treated as indicated (n = 7). Data represent the mean ± SEM. *P < 0.01; #P < 0.01 (one-way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: Control, Staining

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 4. Effect of PGE2 on eNOS phosphorylation in experimental NEC pups. (A) Western blot analysis of total eNOS, phosphorylated eNOS, CD31 and VEGFR2 expression in intestinal homogenates of the pups treated as indicated. The representative bands represent blots of a minimum of three independent experiments. Quantitative analysis of total eNOS (B), the phosphorylated eNOS/eNOS ratio (C), CD31 (D) and VEGFR2 (E) are presented as indicated in triplicate. The histograms are represented as the means ± SEMs. *P < 0.01; +#P < 0.01 (one-way ANOVA); (F) Nitric oxide production was assessed in the intestinal segments of the indicated groups. The eNOS inhibitor L-NAME was used to test the eNOS-dependent mechanism during NO measurement. Columns: means; bars, SEM; *P < 0.01; $P < 0.01, and #P < 0.01 (one-way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 5. eNOS depletion abolished the effect of PGE2 on NEC. (A) Blood bacterial growth quantification under the indicated conditions (n = 5–10 animals per group). (B) Endotoxin levels (LPS) are presented relative to control-fed animals (n = 5–10 animals per group). The concentrations of SIgA (C) and β-defensin-2 (D) were measured in the terminal ileum of mice treated as indicated (n = 5–10 animals per group). The histograms are represented as the means ± SEMs. *P < 0.01 vs. WT control; #,$P < 0.01 vs. the corresponding WT mice with NEC; §P < 0.01 vs. the corresponding eNOS−/− mice with NEC (one-way ANOVA); (E) Representative terminal ileums with H&E staining for the pups from three independent replicate coverslips with experimental NEC treated as indicated. Scale bars: 100 μm; (F) NEC severity scores based on morphological performances were measured by the pathologist (n = 10–18 mice per group). Columns, means; bars, SEM. *P < 0.01 vs. the corresponding WT control; #,$P < 0.01 vs. the corresponding WT mice with NEC; §P < 0.01 vs. the corresponding eNOS−/− mice with NEC (one-way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: Control, Staining

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 7. In vitro assessments of endothelial cell viability modulated by the PGE2 pathway. (A) VEGF levels in the conditioned media of MIMECs were determined by ELISA. MIMECs were treated as indicated, including PGE2, PGE2 and specific pharmacological inhibitors of EP1 (EP1i, SC 51,322), EP2 (EP2i, PF 04,418,948), EP3 (EP3i, L-798, 106), or EP4 (EP4i, L-161, 982). Columns, means; bars, SEM. *P < 0.01 (one-way ANOVA). (B) Cell proliferation was assessed in MIMECs treated as indicated (n = 6). Columns: means of at least three independent repeat experiments; bars, SEM; *P < 0.01; #P < 0.01 (one-way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 6. PGE2 promotes intestinal endothelial cell proliferation. (A) Confocal staining of BrdU (red‒purple) and CD31 (green) was performed to trace endothelial cell proliferation in ileum villi samples from pups treated as indicated. Representative immunofluorescent images are presented. Scale bar, 50 μm; (B) Quantitative endothelial cell measurement based on confocal immunofluorescence staining (5–8 fields/sample, n = 5/group). Columns: means; bars, SEM; *P < 0.01; $P < 0.01; #P < 0.01 (one-way ANOVA). The mRNA expression of CD31 (C) and VEGFR2 (D) with respect to β-actin was assessed in at least three independent experiments in intestinal homogenates from the indicated treatment groups (n = 6). Columns: means; bars, SEM; *P < 0.01; #P < 0.01 (one- way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: Staining, Immunofluorescence, Expressing

Journal: Scientific reports

Article Title: The severity of NEC is ameliorated by prostaglandin E2 through regulating intestinal microcirculation.

doi: 10.1038/s41598-023-39251-x

Figure Lengend Snippet: Figure 8. PGE2 modulates eNOS phosphorylation through the EP4 receptor in MIMECs. (A) Western blot analysis was performed to explore total eNOS and phosphorylated proteins in MIMECs treated as indicated. The blots are representative Western blots of at least three independent experiments. Quantitative total eNOS and phosphorylation expression data (B) were calculated regarding the corresponding loading control (n = 3). The data are presented as the means ± SEMs; *P < 0.01; $P < 0.01; #P < 0.01 (one-way ANOVA).

Article Snippet: For pharmacological PGE2 management and inhibition of various PGE2 receptors, the culture system was further cultured with the following reagents and final concentrations: stabilized PGE2 analog 16,16-dimethyl PGE2 (dmPGE2, 1 μM, R&D Systems), EP1 inhibitor (10 μM, SC 51322; R&D Systems), EP2 inhibitor (10 μM, PF 04418948; R&D Systems), EP3 inhibitor (10 μM, L-798,106; R&D Systems), and EP4 inhibitor (10 μM, L-161,982, R&D Systems).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control

Journal: Cancer Research

Article Title: Prostaglandin E2 Stimulates Human Lung Carcinoma Cell Growth through Induction of Integrin-Linked Kinase: The Involvement of EP4 and Sp1

doi: 10.1158/0008-5472.can-08-2677

Figure Lengend Snippet: Figure 4. PGE2 increased ILK promoter activity via induction of Sp1 in human lung carcinoma cells. A, the human ILK wild-type and mutation promoter construct schematics are presented. These regions contain several transcription factor binding sites, including AP-2, Sp1, and NF-nB. B and C, H1838 cells (1 105) were transfected with a wild-type or two human ILK promoter mutation reporter constructs (shown in A) ligated to luciferase reporter gene and an internal control phRL-TK Synthetic Renilla Luciferase Reporter Vector as described in Materials and Methods for 24 h and then treated as indicated with vehicle control (Con) and dmPGE2 (0.1 Amol/L) for an additional 24 h. The ratio of firefly luciferase to Renilla luciferase activity was quantified as described in Materials and Methods. Columns, mean of at least four independent experiments for each condition; bars, SD. *, significant increase of activity compared with controls. D to F, oligonucleotides containing the Sp1 (D), NF-nB (E), and AP-2 (F) sites were end labeled with [g-32P]ATP and incubated with nuclear extracts (5 Ag) from H1838 cells treated with 0.1 Amol/L dmPGE2 for an additional 24 h. G, oligonucleotides containing Sp1 sites were end labeled with [g-32P]ATP and incubated with nuclear extracts (5 Ag) and Sp1 and Sp2 antibodies (2 Ag/AL each) for 24 h. For competition assays, a molar excess (100) of consensus Sp1 (Cold Sp1) or NF-nB (Cold NF-jB) or AP-2 (Cold AP-2) oligonucleotide was added to the binding reaction. Oligonucleotides containing a mutated Sp1 (Mut Sp1) or NF-nB (Mut NF-jB) or AP-2 (Mut AP-2) site that were end labeled with [g-32P]ATP were used to confirm the binding specificity.

Article Snippet: Polyclonal antibody against Sp1 and Sp2 was purchased from Santa Cruz Biotechnology, Inc. 16,16-Dimethyl-PGE2 (dmPGE2), AH6809, NS398, polyclonal antibodies against EP4, and PGE2 EIA kit were obtained from Cayman Chemical Co. AH23848, Sp1 inhibitor, mithramycin A, and all other chemicals were purchased from Sigma unless otherwise indicated.

Techniques: Activity Assay, Mutagenesis, Construct, Binding Assay, Transfection, Luciferase, Control, Plasmid Preparation, Labeling, Incubation