Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Prostaglandin EP4 Selective Agonist AKDS001 Enhances New Bone Formation by Minimodeling in a Rat Heterotopic Xenograft Model of Human Bone

doi: 10.3389/fbioe.2022.845716

Figure Lengend Snippet: Histological images of human bone and extracted samples. (A) Representative images of hematoxylin and eosin (HE) staining, and anti-EP4 and IgG control immunostaining of human vertebral bone. Arrowheads indicate representative anti-EP4 immunostaining-positive osteoblasts. B, Bone; BM, bone marrow. Scale bars, 1 mm (low magnification), 50 μm (medium magnification), and 50 μm (high magnification). (B) Representative images of HE staining and anti-human vimentin, anti-rat osteocalcin, anti-human osteocalcin immunostaining. Boundary lines of new bone are drawn on HE staining. Arrowheads indicate representative immunostaining-positive cells. XB, xenografted bone; NB, new bone; BM, bone marrow (stroma between xenografted bone). Scale bars, 500 μm (low magnification) and 200 μm (high magnification).

Article Snippet: Hematoxylin and eosin (HE) staining, EP4 (Novus Biologicals, NLS3890), CD31 (Abcam, ab182981), human-vimentin (Abcam, ab16700), human-osteocalcin (Takara Bio, M184), and rat-osteocalcin (Takara Bio, M186) immunostaining were performed with the standard protocols.

Techniques: Staining, Control, Immunostaining

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: a , Immunofluorescence of NeuN and PGE2 receptors in the cerebral cortex showing neuronal expression of Ptger1, Ptger2, Ptger3 and Ptger4 in the adult mouse brain. Scale bar: 50 μm. Same staining was repeated independently in more than 3 mice with similar results. b , Immunofluorescence showing expression of Ptger1, Ptger2, Ptger3 and Ptger4 in primary neuronal cultures (3 weeks in vitro). Ptger2 is mainly located in the neuronal cell body; Ptger1, Ptger3 and Ptger4 are located in both the neuronal cell body and neuronal processes. Scale bar: 50 μm. Same staining was repeated independently in more than 3 batches of primary neuron preparations with similar results.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Immunofluorescence, Expressing, Staining, In Vitro

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: a , b , Live-cell imaging ( a ) and quantitative analysis ( b ) of chronically prion-infected HovS cells expressing control (Ctrl) transgene or one of the four PGE2 receptors (Ptger1–4). Effects of PGE2 treatment on prion-induced cell toxicity were measured with the ratio of GFP signals under the PGE2 condition against the DMSO condition; n = 4 independent experiments. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons: P < 0.0001 (Ptger1 versus Ctrl); P = 0.2246 (Ptger2 versus Ctrl); P = 0.3351 (Ptger3 versus Ctrl); P < 0.0001 (Ptger4 versus Ctrl). c , Immunofluorescence of NeuN, Map2 and Tau showing cellular damage of prion-infected primary neurons treated with different concentrations of Ptger4 agonist L902688. d , Quantification of neuronal density as well as Map2-positive and Tau positive areas shown in c ; n = 6 independent experiments. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.0150 (1 μM versus 0 μM); P < 0.0001 (5 μM versus 0 μM); P < 0.0001 (10 μM versus 0 μM). Map2: P = 0.0020 (1 μM versus 0 μM); P < 0.0001 (5 μM versus 0 μM); P < 0.0001 (10 μM versus 0 μM). Tau: P = 0.0005 (1 μM versus 0 μM); P < 0.0001 (5 μM versus 0 μM); P < 0.0001 (10 μM versus 0 μM). e , f , NeuN immunofluorescence ( e ) and quantification ( f ) showing concentration-dependent enhancement of prion-induced neurodegeneration in L902688-treated Tga20 COCS; n = 12 slices per condition for NBH; prion: 15 slices for 0 μM and 1 μM; 14 slices for 5 μM. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons: P = 0.0810 (NBH + 0 μM versus NBH + 1 μM); P < 0.0001 (NBH + 0 μM versus NBH + 5 μM); P < 0.0001 (NBH + 0 μM versus prion + 0 μM); P < 0.0001 (prion + 0 μM versus prion + 1 μM); P < 0.0001 (prion + 0 μM versus prion + 5 μM).

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Live Cell Imaging, Infection, Expressing, Control, Immunofluorescence, Concentration Assay

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: a , Immunofluorescence of NeuN, Map2 and Tau showing cellular damage of primary neurons treated with high concentration of Ptger4 agonist L902688. b , Quantification of neuronal density as well as Map2 positive and Tau positive areas shown in a . n = 6 independent experiments. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.0001 (NeuN); P < 0.0001 (Map2); P < 0.0001 (Tau). c , Immunofluorescence of NeuN, Map2 and Tau showing no damage of prion-infected primary neurons treated with different concentrations of Ptger1 agonist 17-pt-PGE2. d , Quantification of neuronal density as well as Map2 positive and Tau positive areas shown in c . n = 6 independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.9792 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. Map2: P = 0.9445 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. Tau: P = 0.9165 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. e - f , NeuN immunofluorescence ( e ) and quantification ( f ) showing no change of prion-induced neurodegeneration in 17-pt-PGE2-treated Tga20 COCS. n = 10 slices/condition. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons: P = 0.9799 for NBH + 0 μM vs. NBH + 1 μM, NBH + 0 μM vs. NBH + 5 μM, Prion+0 μM vs. Prion+1 μM, and Prion+0 μM vs. Prion+5 μM. P < 0.0001 for NBH + 0 μM vs. Prion+0 μM.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Immunofluorescence, Concentration Assay, Infection

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: In prion diseases, microglia become activated, and upregulate the pathway responsible for PGE2 biosynthesis, which promotes prion-induced neurodegeneration through binding to neuronal EP4 receptor. NG2 glia serve as a brake in this process, inhibiting microglial Cox2–Ptges pathway and PGE2 biosynthesis through multiple mechanisms (for example, secreted signaling, ECM-receptor interaction and cell–cell contact). Several NG2-glia-derived factors playing a role in this process, such as Tgfb2, Pleiotrophin (Ptn) and Midkine (Mdk), are highlighted in the diagram.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Binding Assay, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2

doi: 10.1084/jem.20180649

Figure Lengend Snippet: Loss of IL-10 signaling leads to overexpression of genes involved in the PGE2 pathway. (A) A simplified schematic diagram of the arachidonic acid pathway from the Kyoto Encyclopedia of Genes and Genomes database showing key steps in the generation of different classes of eicosanoids with a specific focus on the PGE2 synthesis pathway and its receptors. (B) Heat map showing average expression values of genes involved in PGE2 synthesis and its receptors (lower panel) and key rate-limiting genes involved in the synthesis of other eicosanoids (upper panel) in IL-10RB −/− ( n = 3) and control Mφs ( n = 4) after different treatments. (C) Relative mRNA expression for genes involved in PGE2 synthesis (PTGS2 and PTGES) and PGE2 receptors (PTGER2 and PTGER4) between IL-10RA −/− Mφs and isogenic control kolf_2 Mφs with or without LPS stimulation analyzed by RT-qPCR. Data from at least triplicate wells of each condition are presented as means ± SD and are representative of at least three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The following TaqMan probes were used for the indicated genes: Hs00168754_m1 (PTGER2), Hs00168761_m1 (PTGER4), Hs00153133_m1 (PTGS2), Hs00610420_m1 (PTGES), and Hs99999905_m1 (GAPDH).

Techniques: Over Expression, Expressing, Control, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2

doi: 10.1084/jem.20180649

Figure Lengend Snippet: Excessive PGE2 production limits antimicrobial capacity of IL-10RB −/− Mφs. (A) PGE2 levels measured by ELISA in supernatants from IL-10RB −/− and control kolf_2 Mφs ( n = 4) prestimulated overnight with 20 ng/ml rhIL-10 or unstimulated (Unstim) and further stimulated with 2 ng/ml LPS for 6 h in the presence or absence of IL-10 as appropriate. (B and C) Bacterial survival measured at a 5-h time point by gentamicin protection assay and reported as CFU × 10 3 /ml in IL-10RA −/− Mφs ( n = 4) compared with isogenic control kolf_2 Mφs ( n = 4; B) and in IL-10RB comp Mφs ( n = 4) compared with IL-10RB −/− Mφs ( n = 4; C) prestimulated for 2 h with COX2 inhibitors aspirin (10 µM) or indomethacin (10 µM) or left unstimulated and then infected with 10 MOI of S. Typhimurium SL1344 (pssaG:GFP) in the presence or absence of COX2 inhibitors. (D and E) Bacterial survival as in B and C in IL-10RA −/− Mφs ( n = 4) compared with isogenic control kolf_2 Mφs ( n = 4; D) and in IL-10RB comp Mφs ( n = 4) compared with IL-10RB −/− Mφs ( n = 4; E) stimulated with 20 nM EP2, EP4, and EP2 plus EP4 antagonists or left unstimulated. In these experiments, inhibitors were added after initial bacterial uptake. Data shown in all panels are from at least quadruplicate wells, are presented as means ± SD, and are representative of at least three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test was performed using GraphPad software to assess statistical significance. ***, P < 0.001; *, P < 0.05.

Article Snippet: The following TaqMan probes were used for the indicated genes: Hs00168754_m1 (PTGER2), Hs00168761_m1 (PTGER4), Hs00153133_m1 (PTGS2), Hs00610420_m1 (PTGES), and Hs99999905_m1 (GAPDH).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Infection, Software

Journal: PLoS ONE

Article Title: Can the antral follicular count modulate the gene expression of bovine oviducts in Aberdeen Angus and Nelore heifers?

doi: 10.1371/journal.pone.0202017

Figure Lengend Snippet: Genes analyzed in bovine oviducts using TLDA ® system (Gene symbols, gene full name, Applied Biosystems ™ ID, function/reason for selection, and reference).

Article Snippet: PTGER4 , Prostaglandin E receptor 4 , Bt03223849_m1 , Gamete transport , [ , ] .

Techniques: TLDA Assay, Selection, Control

Journal: PLoS ONE

Article Title: Can the antral follicular count modulate the gene expression of bovine oviducts in Aberdeen Angus and Nelore heifers?

doi: 10.1371/journal.pone.0202017

Figure Lengend Snippet: HSPA5 , FUCA2 , ANXA2 , VEGFA , and PTGER4 (mean ± SEM) in ipsilateral and contralateral oviducts (n = 16 animals). Expression of target genes were normalized by reference genes using 2(-ΔΔCt). Different letters indicate significant difference (p ≤ 0.05) between means.

Article Snippet: PTGER4 , Prostaglandin E receptor 4 , Bt03223849_m1 , Gamete transport , [ , ] .

Techniques: Expressing

Journal: Cancers

Article Title: Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer

doi: 10.3390/cancers12082008

Figure Lengend Snippet: NFκB1 , COX-2 , EP4 , and VEGFA gene expression: ‘N’ indicates normoxia, and ‘H’ indicates hypoxia. ( A ) NFκB1 , ( B ) COX-2 , ( C ) EP4 , and ( D ) VEGFA represents gene expression in MCF7, MCF7-miR526b, and MCF7-miR655 cell lines. ( E ) NFκB1 , ( F ) COX-2 , ( G ) EP4 , and ( H ) VEGFA gene expression in SKBR3-miR526b and MCF7-COX2 cell lines. Data are presented as the mean ± SEM of quadruplicate replicates; * p < 0.05, ** p < 0.01 and *** p < 0.001. x indicates p < 0.05, xx indicates p < 0.01 and xxx indicates p < 0.001. * Also indicates comparison between normoxia and hypoxia of the same cell line and x indicates comparison between cell lines only in hypoxia.

Article Snippet: The expressions of pri-miR526b (Hs03296227_pri), pri-miR655 (Hs03304873_pri), hsa-miR-526b-5p (assay ID #002382), hsa-miR-655-3p (assay ID #001612), HIF-1α (Hs00153153_m1), VEGFA (Hs00900055_m1), COX-2 (Hs00153133_m1), EP4 (Hs00964382_g1), VHL (Hs03046964_s1), NFκB1 (Hs00231653_m1), TWIST1 (Hs04989912_s1), VIM (Hs00185584_m1), SNAIL (Hs00195591_m1), CDH1 (Hs00170423_m1), TXNRD1 (Hs00917067_m1), SALL4 (Hs01010838_g1), ZNF207 (Hs01045973_m1), and NR2C2 (Hs00991824_m1) were quantified using relative gene expression analysis.

Techniques: Gene Expression, Comparison

Journal: Cancers

Article Title: Chemically Induced Hypoxia Enhances miRNA Functions in Breast Cancer

doi: 10.3390/cancers12082008

Figure Lengend Snippet: Linking COX-2, EP4, and PI3K/Akt pathways with hypoxia, miR526b, and miR655: red lines indicate functions induced by miR526b and miR655, while black lines indicate functions that are not directly induced by miRNAs. Arrows indicate induction, while T-shaped lines indicate inhibition.

Article Snippet: The expressions of pri-miR526b (Hs03296227_pri), pri-miR655 (Hs03304873_pri), hsa-miR-526b-5p (assay ID #002382), hsa-miR-655-3p (assay ID #001612), HIF-1α (Hs00153153_m1), VEGFA (Hs00900055_m1), COX-2 (Hs00153133_m1), EP4 (Hs00964382_g1), VHL (Hs03046964_s1), NFκB1 (Hs00231653_m1), TWIST1 (Hs04989912_s1), VIM (Hs00185584_m1), SNAIL (Hs00195591_m1), CDH1 (Hs00170423_m1), TXNRD1 (Hs00917067_m1), SALL4 (Hs01010838_g1), ZNF207 (Hs01045973_m1), and NR2C2 (Hs00991824_m1) were quantified using relative gene expression analysis.

Techniques: Inhibition