Journal: PLoS ONE

Article Title: Comparison of Angiogenic, Cytoprotective, and Immunosuppressive Properties of Human Amnion- and Chorion-Derived Mesenchymal Stem Cells

doi: 10.1371/journal.pone.0088319

Figure Lengend Snippet: (A) Inhibition of human CD4+ T cell proliferation upon co-culture with human amnion, chorion, and bone marrow MSCs. (B) The concentration of PGE2 in FM-MSC-conditioned medium was measured by ELISA. Amnion MSCs secreted a significant amount of PGE2 compared with chorion MSCs. (C, D) Effect of human amnion (C) or chorion (D) MSC transplantation in a murine GVHD model. Treatment with amnion MSCs significantly reduced recipient weight loss in a mouse model of GVHD. *p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: The concentrations of the following growth factors were measured using ELISA kits: hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE2), according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Techniques: Inhibition, Co-Culture Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transplantation Assay

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Structure of the binding interface between the UFD of S. pombe Uba1 (interacting residues colored pink) and the alpha-1 helix (aa 1–18, colored by amino acid property) of the E2 Ubc15 (cyan), as revealed by the x-ray structure of the S. pombe Uba1/Ubc15 complex (PDB: 5KNL) . ( b ) Amino acid sequences of the SAH-Ubc15 E7R staple-scanning library, which was generated by inserting all-hydrocarbon i, i+4 or i, i+7 staples sequentially along the length of the Ubc15 E7R peptide. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine (replacement for methionine, whose sulfur residue decreases the efficiency of ruthenium-catalyzed olefin metathesis). ( c ) Helical wheel depiction of SAH-Ubc15 E7R peptides and their staple positions (helical residues span from aa 5–18). ( d ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by the indicated SAH-UBC15 E7R stapled peptides. Thioester transfer activity was monitored by the shift in E2 molecular weight from 17 kDa (free UBE2D2) to 25 kDa (UBE2D2~ubiquitin conjugate), as detected by gel electrophoresis and silver stain. Vehicle lane contained 1.5% DMSO and control lane lacked UBE1 protein. The bar graph represents mean inhibition relative to vehicle control ± s.e.m., as quantified and averaged across three independent biological replicates. Uncropped gel for panel d is available online. Source data for the thioester transfer assay plot is available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Binding Assay, Generated, Residue, Inhibition, Ubiquitin Proteomics, Activity Assay, Molecular Weight, Nucleic Acid Electrophoresis, Silver Staining, Control

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: ( a ) Amino acid sequences of stapled peptides corresponding to the N terminus and alpha-1 helix of UBE2D2 (aa 1–16), UBE2G2 (aa 1–14), and UBE2A (aa 1–17), which incorporate the optimal staple position identified for SAH-Ubc15–11. X, S5-pentenyl alanine; 8, R5-octenyl alanine; B, norleucine. ( b ) Comparative dose-responsive inhibition of UBE1 by the indicated SAH-E2 h1 peptides, as monitored by thioester transfer assay, gel electrophoresis, and silver stain. ( c ) Quantitation of the data in b by ImageJ analysis. Data are mean percent inhibition ± s.e.m. for three independent biological replicates. ( d ) Circular dichroism spectra of UBE2A (BSTPARRRLBRDFKRLQ, where B is norleucine) and SAH-UBE2A, demonstrating induction of α-helicity by insertion of the i, i+7 staple. ( e ) Inhibition of UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2 by SAH-UBE2A but not the corresponding unmodified template peptide, UBE2A. Data are mean inhibition ± s.e.m. for three independent biological replicates. ( f ) Comparative protease resistance of SAH-UBE2A and UBE2A upon treatment with proteinase K, as measured by HPLC analysis. SAH-UBE2A displays a 23-fold longer half-life than UBE2A. Data are mean ± s.d. for experiments performed in triplicate. ( g ) SAH-UBE2A but not UBE2A was significantly protected from deuterium exchange upon incubation with UBE1. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for UBE2A or SAH-UBE2A in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. ( h ) C-terminally biotinylated SAH-UBE2A directly bound to recombinant human UBE1 (MW 118 kDa), as demonstrated by streptavidin capture, gel electrophoresis, and silver stain. ( i ) C-terminally FITCylated SAH-UBE2A bound to UBE1 with a K d of 384 ± 43 nM, as assessed by fluorescence polarization assay. Error bars are s.d. for assays conducted in technical triplicate and performed three times with independent preparations of peptide and protein (all 9 points for each dosing level are plotted). Uncropped gels for panels b , e , and h are available online. Source data for thioester transfer assays, CD spectra, protease resistance testing, HXMS analyses, and FP plot are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Inhibition, Nucleic Acid Electrophoresis, Silver Staining, Quantitation Assay, Circular Dichroism, Ubiquitin Proteomics, Incubation, Labeling, Recombinant, Fluorescence

Journal: Nature chemical biology

Article Title: Targeting a Helix-in-Groove Interaction Between E1 and E2 Blocks Ubiquitin Transfer

doi: 10.1038/s41589-020-0625-7

Figure Lengend Snippet: (a) Sequence compositions of the unmodified UBE2A template peptide and stapled constructs SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A . (b) Comparative effects of UBE2A, SAH-UBE2A, SAH-UBE2A R7E , and SAH-UBE2A R7A/L9A/B10A on UBE1-mediated thioester transfer of ubiquitin to the E2 enzyme UBE2D2. Whereas SAH-UBE2A (10 μM) completely inhibits UBE1 activity, triple mutagenesis abrogates the inhibitory effect and single R7E mutagenesis has an intermediate effect. (c) Progressive loss of UBE1 protection from deuterium exchange into SAH-UBE2A upon single R7E and triple R7A/L9A/B10A mutagenesis. The relative difference in deuterium uptake after 10 sec of labeling is shown. The difference in uptake was calculated from the mean deuterium level for each SAH-UBE2A peptide in the presence of UBE1 minus that of the peptide alone. Mean deuterium levels were obtained from at least two independent biological replicates of each condition. (d) Difference in deuterium uptake plots demonstrate the relative deuterium incorporation of SAH-UBE2A/UBE1, SAH-UBE2A R7E /UBE1, or SAH-UBE2A R7A/L9A/B10A /UBE1 minus the relative deuterium incorporation of UBE1 at 10 min of deuterium labeling. The shaded gray region indicates differences in deuteration that are below the meaningful differences threshold. Consistent with results of both the thioester transfer assay (b) and peptide deuterium exchange (c), and the predicted SAH-UBE2A/UBE1 binding mode, triple mutagenesis essentially abrogates the effect of SAH-UBE2A on UBE1 conformation, with single R7E mutagenesis having an intermediate influence. Data are representative of three independent replicates for SAH-UBE2A, and two independent replicates for SAH-UBE2A R7E and SAH-UBE2A R7A/L9A/B10A . Each peptide, from N- to C-terminus, was given a peptide number to simplify the presentation. The peptide list and residue identity of each peptide can be found in . Uncropped gel for panel b is available online. Source data for the HXMS analyses are available online.

Article Snippet: For UBA3 thioester transfer assays, 15 nM NAE1/UBA3 was substituted for UBE1 (Boston Biochem E-313), 135 nM UBE2M for UBE2D2 (Boston Biochem E2–656), and 1 μM NEDD8 (Boston Biochem UL-812) for ubiquitin.

Techniques: Sequencing, Construct, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Labeling, Binding Assay, Residue

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