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Figure Lengend Snippet: Simplified schematic illustration of pathways for Wnt/β-catenin, ERK/MAPK and PI3K/Akt and PGE 2 -metabolism. A) Canonical Wnt/β-catenin signaling. The engagement of the Wnt receptor, Frizzled, leads to the inhibition of the β-catenin destruction complex, composed of APC, axin and GSK3β. β-catenin thereby avoids ubiquitination and subsequent degradation, thus allowing it to translocate to the nucleus to activate an array of regulatory genes. B) The RAS/RAF/MEK/ERK MAPK pathway. Stimulation of the receptor tyrosine kinase (RTK) or G-protein coupled receptors (GPCRs) leads to sequential activation of RAS, RAF, MEK, and ERK causing modification of substrates promoting cell survival and proliferation. C) In the PI3K/Akt pathway, activation of the RTK or GPCRs leads to sequential modification of phosphatidyl inositol residues of the phospholipid bilayer. In this process, PI3K generates PIP3. PIP3 in association with PDK1 activates Akt. Akt then modulates the activity of downstream substrates including mTOR, thus promoting proliferation and cell survival. D) PGE 2 -metabolism. PGE 2 -synthesis begins with catalytic hydrolysis of membrane phospholipids by cytoplasmic phospholipase A2 (cPLA 2 ), thus releasing arachidonic acid (AA). By the action of the COX-1 and COX-2, AA is converted to prostaglandin H 2 (PGH 2 ). PGH 2 is then converted to PGE 2 by prostaglandin E synthase (PTGES). The main exporter of PGE 2 is thought to be multi-drug resistance related polypeptide 4 (MRP4). Removal of PGE 2 from the extracellular compartment around target cells occurs by diffusion to the blood stream and subsequent uptake and degradation in lung, liver or kidney endothelial cells or by import to colonic epithelial cells through the prostaglandin transporter (PGT) and subsequent degradation by 15-prostaglandin dehydrogenase (15-PGDH). Through autocrine and paracrine signaling, extracellular PGE 2 stimulates the prostaglandin receptors EP1–4. The EPs are GPCRs with EP1 being Gαq-coupled while EP2 and EP4 are Gαs-coupled. EP3 is capable of coupling with different G-proteins including Gαi, Gαs and Gαq

Article Snippet: The following antibodies were tested: Mouse anti-EP3 antibody (5F5), sc-57,105 (Santa Cruz Biotechnology), Rabbit anti-EP3 antibody, 101,760 (Cayman Chemicals, Ann Arbor, MI, USA), Rabbit anti-EP3 antibody, APR-065 (Alomone Labs, Jerusalem, Israel), Rabbit anti-EP3 antibody (PTGER3), NBP1–84835 (Bio-techne, Abingdon, United Kingdom).

Techniques: Inhibition, Activation Assay, Modification, Activity Assay, Diffusion-based Assay

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Figure Lengend Snippet: Quantitative real-time PCR primer sequences

Article Snippet: The following antibodies were tested: Mouse anti-EP3 antibody (5F5), sc-57,105 (Santa Cruz Biotechnology), Rabbit anti-EP3 antibody, 101,760 (Cayman Chemicals, Ann Arbor, MI, USA), Rabbit anti-EP3 antibody, APR-065 (Alomone Labs, Jerusalem, Israel), Rabbit anti-EP3 antibody (PTGER3), NBP1–84835 (Bio-techne, Abingdon, United Kingdom).

Techniques: Real-time Polymerase Chain Reaction

Journal:

Article Title:

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Figure Lengend Snippet: Difference in mRNA expression in CRN versus controls

Article Snippet: The following antibodies were tested: Mouse anti-EP3 antibody (5F5), sc-57,105 (Santa Cruz Biotechnology), Rabbit anti-EP3 antibody, 101,760 (Cayman Chemicals, Ann Arbor, MI, USA), Rabbit anti-EP3 antibody, APR-065 (Alomone Labs, Jerusalem, Israel), Rabbit anti-EP3 antibody (PTGER3), NBP1–84835 (Bio-techne, Abingdon, United Kingdom).

Techniques: Expressing

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: Fig. 7 | PGE2 enhances prion neurotoxicity mainly through the EP4 receptor (Ptger4). 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

Article Snippet: 3 n atu re p o rtfo lio | rep o rtin g su m m ary A p ril 2 0 2 3 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used mouse monoclonal antibody against actin (1:10,000, Merck Millipore, MAB1501R, clone C4); mouse monoclonal antibody against PrP (POM1, 1:5000, homemade); rabbit polyclonal antibody against NG2 (1:500, MERCK, AB5320); rabbit polyclonal antibody against PDGFRα (1:500, Santa Cruz, SC-338); rabbit monoclonal antibody against NeuN (1:1000, Abcam, ab177487, clone EPR12763); rabbit polyclonal antibody against NG2 (1:500, a gift from Prof. Stallcup); rabbit polyclonal antibody against Iba1 (1:500, Wako, 019-19741); Rat monoclonal antibody against Cd68 (1:200, BioRad, MCA1957, clone FA-11); rabbit polyclonal antibody against Map2 (1:200, Biolegend, 840601), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, sc-166475, clone D-12); mouse monoclonal antibody against Ptges (1:200, Santa Cruz, sc-365844, clone H-3); rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, BS-6316R); rabbit monoclonal antibody against EP2 (1:200, Abcam, ab167171, clone EPR8030(B)); rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, 101760); mouse monoclonal antibody against EP4 (1:200, ProteinTech, 66921-1-Ig, clone 4A2A12); rat monoclonal antibody against CD11b antibody (30 ul in 10 ml, ThermoFisher Scientific, 14-0112-82, clone M1/70), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, MN1010, clone BT2), chicken polyclonal antibody against NeuN (1:1000, Merck, ABN91), goat polyclonal antibody against rat IgG (30 ul in 10 m, Jackson ImmunoResearch, 112-005-167); HRP-conjugated goat antirabbit IgG antibody (1:10,000, Jackson ImmunoResearch, 111-035-003); HRP-conjugated goat anti-mouse IgG antibody (1:10,000, Jackson ImmunoResearch, 115-035-003); Alexa488-conjugated goat anti-mouse IgG antibody (1:3000, ThermoFisher Scientific, A32723); Alexa594-conjugated goat anti-rabbit IgG antibody (1:3000, ThermoFisher Scientific, A32740); Alexa647-conjugated goat anti-chicken IgG antibody (1:3000, ThermoFisher Scientific, A32933).

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

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: EP3 is upregulated in cerebral small arteries of RHRsp and deletion of EP3 does not affect BP. (A) Immunofluorescence for EP3 (Green) and αSMA (Red) of SD and RHRsp. The cerebral small arteries (10-65 μm) are indicated by the immunostaining of αSMA. Bar, 50 µm. (B) Blood pressure in SD and RHRsp. EP3, E prostanoid 3; SD, Sprague Dawley rat; RHRsp, Stroke-prone renovascular hypertensive rat; αSMA, smooth muscle actin alpha; BP, blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure; MBP, mean blood pressure; m, month; **, p<0.01; ****, p<0.0001.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Immunofluorescence, Immunostaining

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: The deposition of collagen IV in the vessel wall of cerebral small arteries. Immunofluorescence for αSMA (Green) and collagen IV (Red) of the animals. The cerebral small arteries (10-65 μm) are indicated by the immunostaining of αSMA. Bar, 50 µm. EP3, E prostanoid 3; RHRsp, Stroke-prone renovascular hypertensive rat; αSMA, smooth muscle actin alpha, *p<0.05, ***p<0.001, ****p<0.0001.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Immunofluorescence, Immunostaining

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: The deposition of laminin in the vessel wall of cerebral small arteries. Immunofluorescence for αSMA (Green) and laminin (Red) of the animals. The cerebral small arteries (10-65 μm) are indicated by the immunostaining of αSMA. Bar, 50 µm. EP3, E prostanoid 3; RHRsp, Stroke-prone renovascular hypertensive rat; αSMA, smooth muscle actin alpha, *p<0.05, ***p<0.001.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Immunofluorescence, Immunostaining

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: The deposition of collagen I in the vessel wall of cerebral small arteries. Immunofluorescence for αSMA (Green) and collagen I (Red) of the animals. The cerebral small arteries (10-65 μm) are indicated by the immunostaining of αSMA. Bar, 50 µm. EP3, E prostanoid 3; RHRsp, Stroke-prone renovascular hypertensive rat; αSMA, smooth muscle actin alpha, *p<0.05, **p<0.01.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Immunofluorescence, Immunostaining

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: The deposition of fibronectin in the vessel wall of cerebral small arteries. Immunofluorescence for αSMA (Green) and fibronectin (Red) of the animals. The cerebral small arteries (10-65 μm) are indicated by the immunostaining of αSMA. Bar, 50 µm. EP3, E prostanoid 3; RHRsp, Stroke-prone renovascular hypertensive rat; αSMA, smooth muscle actin alpha, *p<0.05.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Immunofluorescence, Immunostaining

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: Change of extracellular matrix expression in rat BMVSMCs under different stimulation. (A) Representative western blots and quantification of the expression of fibronectin, laminin, collagen I, and collagen IV in rat BMVSMCs under different stimulation. (B) Quantification of western blots of fibronectin, laminin, collagen I, and collagen IV. EP3, E prostanoid 3; BMVSMCs, brain microvascular smooth muscle cells; ANGII, Angiotensin II. *, p<0.05; **, p<0.01; ****, p<0.0001.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Expressing, Western Blot

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: Quantification of western analysis. (A-B) TGF-β1 is elevated in rat BMVSMCs under ANGII stimulation. Suppressing TGF-β1 by LY364947 reverses the high expression of ECM in rat BMVSMCs under ANGII stimulation. (C, E) Deletion of EP3 rescues ANGII-induced activation of TGF-β1/ SMAD2/signaling. (D, F) The downregulation of ECM by the inhibition of EP3 is abolished by pretreatment with PTX in BMVSMCs. EP3, E prostanoid 3; BMVSMCs, brain microvascular smooth muscle cells; ECM, extracellular matrix; TGF-β1, transforming growth factor-beta1; ANGII,Angiotensin II. *, p<0.05; **, p<0.01; ***, p<0.001; NS, no significant statistical difference.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: Western Blot, Expressing, Activation Assay, Inhibition

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: Deletion of EP3 attenuates reduced CBF in RHRsp. (A) Quantitative results of CBF. (B) Representative picture of CBF. EP3, E prostanoid 3; CBF, cerebral blood flow; RHRsp, Stroke-prone renovascular hypertensive rat; H, high; L, low; *, p<0.05, **, p<0.01.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques:

Journal: Aging and Disease

Article Title: EP3 Receptor Deficiency Improves Vascular Remodeling and Cognitive Impairment in Cerebral Small Vessel Disease

doi: 10.14336/AD.2021.0627

Figure Lengend Snippet: Deletion of EP3 attenuates cognitive impairment in RHRsp. (A) Morris water maze test: escape latency in place navigation test. (B) Morris water maze test: time spent in the target quadrant in the spatial probe test. (C) Morris water maze test: the number of times crossing the platform in the spatial probe test. EP3, E prostanoid 3; RHRsp, Stroke-prone renovascular hypertensive rat; **, p<0.01; ***, p<0.001.

Article Snippet: EP3 -/- (knockout) rats on a Sprague-Dawley background were created by Beijing Biocytogen (Beijing, China) using CRISPR/Cas9-based technology.

Techniques: