Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 1. Deletion of the CM-EP4 improves ISO-induced cardiac diastolic dysfunction and fibrosis. A) The experimental scheme showing the generation of EP4f/f-𝛼-MyHCCre+ (CM-EP4−/−) mice and ISO-induced cardiac fibrosis model. The EP4f/f-𝛼-MyHCCre+ (CM-EP4−/−) mice and control mice (EP4f/f) were generated by crossing the EP4f/f mice with the 𝛼-MyHCCre+ mice. Male mice aged 8–10 weeks were administered subcutaneous injections of ISO daily for 7 consecutive days to induce cardiac fibrosis. B&C. Representative images of peak velocity flow in early diastole (E, m/s) to peak velocity flow in late diastole by atrial contraction (A, m/s) in the EP4f/f and EP4f/f-𝛼-MyHCCre+ mice injected with saline (top panels) or ISO (bottom panels) for 7 days (B). The ratio of E/A was calculated (C). Transverse scale bar = 100 ms. Vertical scale bar = 0.4 m s−1. n = 4–9 mice per group. D&E. Representative images of Masson’s trichrome staining of the hearts of the EP4f/f and EP4f/f-𝛼-MyHCCre+ mice receiving saline (left panels) or ISO (right

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: Control, Generated, Injection, Saline, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 2. Deletion of the CF-EP4 ameliorates ISO-induced left ventricular diastolic dysfunction and fibrosis. A. The experimental scheme showing the generation of EP4f/f-S100A4Cre+ (CF-EP4−/−) mice and ISO-induced cardiac fibrosis model. The EP4f/f-S100A4Cre+ mice and control mice (EP4f/f) were generated by crossing the EP4f/f mice and S100A4Cre+ mice. Male mice aged 8–10 weeks were subjected to subcutaneous injections of ISO for 7 days to induce cardiac fibrosis. B,C) Representative images of peak velocity flow in early diastole (E, m/s) to peak velocity flow in late diastole by atrial contraction (A, m/s) in mice injected with saline (top panels) or ISO (bottom panels) for 7 days (B). The ratio of E/A was calculated to reveal the cardiac diastolic function (C). Transverse scale bar = 100ms. Vertical scale bar = 0.4 m s−1. n = 7–10 mice per group. D,E) Representative images of Masson’s trichrome staining of the hearts of the EP4f/f and EP4f/f-S100A4Cre+ mice treated with saline (left panels) or ISO (right panels) for 7 days. General view,

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: Control, Generated, Injection, Saline, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 3. EP4 promotes ISO-induced TGF-𝛽1 expression in the CMs via the PKA pathway. A) qRT-PCR analysis showing upregulation of the mRNA expression of TGF-𝛽1 in wild-type mouse heart tissues after ISO treatment for 7 days. n = 6 per group. B,C) Representative images of immunohisto- chemical staining of TGF-𝛽1 in wild-type mouse heart tissues after ISO treatment for 7 days (B). Schematic showing of ISO-induced TGF-𝛽1 expression in the CMs (C). Scale bar = 50 μm. D) qRT-PCR analysis of the mRNA expression of TGF-𝛽1 in the hearts of the EP4f/f and EP4f/f-𝛼-MyHCCre+ mice receiving ISO treatment for 7 days. n = 6 per group. E,F) Representative images of immunohistochemical staining of TGF-𝛽1 in the hearts of the 8–10 weeks old male EP4f/f and EP4f/f-𝛼-MyHCCre+ mice receiving saline (left panel) or ISO (right panel) treatment for 7 days. Papillary muscle (PM) area, interstitial area near the endocardium (INE), and perivascular (PV) area were shown, respectively (E). Quantitative analysis of TGF-𝛽1 positive areas was

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: Expressing, Quantitative RT-PCR, Staining, Immunohistochemical staining, Saline

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 4. Blockade of EP4 suppresses TGF-𝛽1 mediated collagen production in the CFs by inhibiting phosphorylation of Smad2/3. A,B) Western blot analysis showing the effect of 24-h TGF-𝛽1 (10 ng mL−1) treatment on collagen I and collagen III expression in primary cultured neonatal rat cardiac fibroblasts (NRCFs) pretreated with or without grapiprant (1μM) for 30 min (A). Quantitative analysis was performed using image J software (B). n = 5. C,D) Western blot analysis demonstrating the effect of 24-h TGF-𝛽1 (10 ng mL−1) treatment on collagen I and collagen III expression in the NRCFs pretreated with or without MF498 (0.1μM) for 30 min (C). Quantitative analysis was performed using image J software (D). n = 3. E,F) Western blot analysis showing the effect of 3-h TGF-𝛽1 (10 ng mL−1) treatment on phosphorylation levels of Smad2 and Smad3 in the NRCFs pretreated with or without grapiprant (1μM) for 30 min (E). Quantitative analysis was performed using image J software (F). n = 5. G,H) Immunoblot analysis demonstrating the effect of 3-h TGF-𝛽1 (10 ng mL−1) treatment on phosphorylation levels of Smad2 and Smad3 in the NRCFs pretreated with or without MF498 (0.1μM) for 30 min (G). Quantitative analysis was performed using image J software (H). n = 5. I,J) Immunofluorescence staining showing the effect of 3-h TGF-𝛽1 (10 ng mL−1) treatment on Smad3 (green) nuclear translocation in the NRCFs pretreated with or without grapiprant (1μM) (I) or MF498 (0.1μM) (J) for 30 min. DAPI (blue) staining showed the nucleus. Scale bar = 50 μm. Data were presented as mean±SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA followed by the Tukey’s multiple comparisons test (B,D,F,H).

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: Phospho-proteomics, Western Blot, Expressing, Cell Culture, Software, Staining, Translocation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 5. EP4 augments the TGF-𝛽1 signaling by the formation of the EP4-TGFBRII complex. A) qRT-PCR analysis demonstrating the effect of 24-h TGF-𝛽1 (10 ng mL−1) treatment on the mRNA expression of TGF-𝛽1 in the NRCFs pretreated with or without grapiprant (1μM) for 30 min. n = 3. B,C) Western blot assay showing the effect of 24-h TGF-𝛽1 (10 ng mL−1) treatment on the protein expression of TGF-𝛽1 in the NRCFs pretreated with or without grapiprant (1μM) for 30 min (B). Quantitative analysis was performed using image J software (C). n = 5. D. ELISA measurement of TGF-𝛽1 levels in the medium of cultured NRCFs. The cells were pretreated with or without grapiprant (1μM) for 30 min and then treated with TGF-𝛽1 (10 ng mL−1) for 24 h. The relative contents of TGF-𝛽1 in the medium were calculated under basal and TGF-𝛽1-treated conditions. n = 5. E–H) The top ten binding modes of the EP4-TGFBRII complex. The left image is the front view and the right image is the top view (E). The snug-fit-in model of last

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Cell Culture, Binding Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 6. Inactivation of EP4 limits CFs proliferation in vivo and in vitro. A,B) Immunofluorescence analysis of the tdTomato-positive CFs in the hearts of the tdTomatof/+-S100A4Cre+ mice receiving ISO treatment for 7days. Heart sections were stained with DAPI to visualize the nuclei. Scale bar = 100 μm (A). Schematic presentation of ISO-induced CF proliferation (B). C,D) Representative images of immunohistochemical staining of PCNA in the hearts of the 8–10 weeks old male EP4f/f and EP4f/f-S100A4Cre+ mice treated with saline (left panel) or ISO (right panel) for 7 days. PM, papillary muscle area; INE, interstitial area near the endocardium (C). Quantitative analysis of the PCNA-positive areas by image J software (D). Scale bar = 20 μm. n = 5–6 per group. E,F) Representative images of immunohistochemical staining of PCNA in the hearts of the 8–10 weeks old male EP4f/f and EP4f/f-𝛼-MyHCCre+

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: In Vivo, In Vitro, Staining, Immunohistochemical staining, Saline, Software

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting the E Prostanoid Receptor EP4 Mitigates Cardiac Fibrosis Induced by β-Adrenergic Activation.

doi: 10.1002/advs.202413324

Figure Lengend Snippet: Figure 7. Double knockout of the EP4 gene in both CMs and CFs improves ISO-induced cardiac diastolic dysfunction and fibrosis. A) The experimental protocol for the generation of the double knockout mice with EP4 deficiency in both CMs and CFs (EP4f/f-DoubleCre+) mice and ISO-induced cardiac fibrosis model. The EP4f/f-DoubleCre+ mice were generated by crossing the EP4f/f-𝛼-MyHCCre+ mice and EP4f/f- S100A4Cre+ mice. The littermate mice without 𝛼-MyHC-Cre or S100A4-Cre (EP4f/f) were used as control mice. Male mice aged 8–10 weeks were subcutaneously injected ISO for 7 days to induce cardiac fibrosis. B) PCR-based genotyping showing the S100A4-Cre positive and 𝛼-MyHC-Cre positive bands. C) Representative immunohistochemical staining showing the absence of the EP4 protein in the hearts of the EP4f/f-DoubleCre+ mice. Scale bar = 20μm. D) Representative photographs showing the hearts of the EP4f/f and EP4f/f-DoubleCre+ mice receiving ISO treatment for 7 days. Scale bar = 2mm. E,F) Representative echocardiography of peak

Article Snippet: The slices were incubated with the primary antibody against EP4 (1:50, Santa Cruz) and followed by incubation with fluorescence-conjugated secondary antibody.

Techniques: Double Knockout, Generated, Control, Injection, Immunohistochemical staining, Staining

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: Reproductive Medicine and Biology

Article Title: Changing prostaglandin E2 (PGE 2 ) signaling during lesional progression and exacerbation of endometriosis by inhibition of PGE 2 receptor EP2 and EP4

doi: 10.1002/rmb2.12426

Figure Lengend Snippet: Summary result of immunohistochemistry and Masson trichrome staining in human samples showing the dynamic changes as endometriotic lesions progress over time. Dynamic changes in the extent of fibrosis in tissues/lesions in different groups of mice (A). Dynamic changes in the immunostaining of COX‐2 the stromal (B) and epithelial (C) components in tissues/lesions in different groups of mice. Dynamic changes in the immunostaining of EP2 the stromal (D) and epithelial (E) components in tissues/lesions in different groups of mice. Dynamic changes in the immunostaining of EP4 the stromal (F) and epithelial (G) components in tissues/lesions in different groups of mice. In all panels, the data are represented by the means ± SDs, and Kruskal's rank test was used. Symbols of statistical significance levels: * P < 0.05; ** P < 0.01; *** P < 0.001; NS: not statistically significant ( P > 0.05)

Article Snippet: Serial 4‐μm sections were obtained from each block, with the subsequent slides for immunohistochemistry (IHC) analysis for cyclooxygenase‐2 (COX‐2, 1:300; #ab15191; Abcam), EP2 (1:300; #ab167171; Abcam), EP4 (1:300; #bs‐8538r; Bioss), and αsmooth muscle actin (α‐SMA, 1:100; #ab5694.

Techniques: Immunohistochemistry, Staining, Immunostaining

Journal: Reproductive Medicine and Biology

Article Title: Changing prostaglandin E2 (PGE 2 ) signaling during lesional progression and exacerbation of endometriosis by inhibition of PGE 2 receptor EP2 and EP4

doi: 10.1002/rmb2.12426

Figure Lengend Snippet: P ‐values for the difference between the lesions of interest and the normal endometrium based on linear regression analysis

Article Snippet: Serial 4‐μm sections were obtained from each block, with the subsequent slides for immunohistochemistry (IHC) analysis for cyclooxygenase‐2 (COX‐2, 1:300; #ab15191; Abcam), EP2 (1:300; #ab167171; Abcam), EP4 (1:300; #bs‐8538r; Bioss), and αsmooth muscle actin (α‐SMA, 1:100; #ab5694.

Techniques:

Journal: Reproductive Medicine and Biology

Article Title: Changing prostaglandin E2 (PGE 2 ) signaling during lesional progression and exacerbation of endometriosis by inhibition of PGE 2 receptor EP2 and EP4

doi: 10.1002/rmb2.12426

Figure Lengend Snippet: Representative photomicrographs of immunostaining and histochemistry analysis (left panel), along with data summary (right panel). On the left panel, different rows show different markers as indicated. Different columns represent different tissue samples from endometriotic lesions taken from control mice, and mice treated with low‐, medium‐, and high dose of EP2 and EP4 inhibitors (see text for more details), respectively. All mice were induced with deep endometriosis. In Masson trichrome staining, the collagen fibers in lesions were stained in blue. In all figures, magnification: ×400. Scale bar = 50 μm. On the right panel, the results are summarized by the boxplot, separated by the stromal or epithelial component, when applicable. The dashed line represents the median value of all mice. All comparison was made in reference to the control group, and Wilcoxon's test was used. Symbols of statistical significance levels: * P < 0.05; ** P < 0.01; *** P < 0.001; NS: not statistically significant ( P > 0.05)

Article Snippet: Serial 4‐μm sections were obtained from each block, with the subsequent slides for immunohistochemistry (IHC) analysis for cyclooxygenase‐2 (COX‐2, 1:300; #ab15191; Abcam), EP2 (1:300; #ab167171; Abcam), EP4 (1:300; #bs‐8538r; Bioss), and αsmooth muscle actin (α‐SMA, 1:100; #ab5694.

Techniques: Immunostaining, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 1 COX-1/2 and COX-2 inhibitors and EP4 antagonist reduce VEGF-C and VEGF-D production in C3L5 cells. (a) Western blot analysis of proteins in C3l5 cell lysates revealed expression of COX-2 and all the EP receptors. (b, c) Both COX1/2 and COX-2 inhibitors as well as the EP4 (but not EP1, EP2 and EP3) antagonist suppressed VEGF-C and -D mRNA levels relative to GAPDH mRNA (presented as a fraction of control vehicle-treated cells) as well as secreted VEGF-C (d, e) measured with ELISA. C3L5 cells express VEGF-C (80 kDa) and VEGF-D (21 kDa) (f, control) measured with western blot, which were reduced by treatments with COX inhibitors and EP4 (but not EP1) antagonist, as compared with vehicle-treated control cells (f, g). siRNA-mediated knockdown of EP4 resulted in significant drop in VEGF-C and VEGF-D mRNA (h). Data presented in (a, b), (c, d) and (e, f) are from three different representative experiments. Data represent means (n ¼ 4)±s.e., *Po0.05, **Po0.01, ***Po0.001.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 2 Therapy with COX-1/2 and COX-2 inhibitors or EP4 antagonist, but not EP1 antagonist reduces primary tumor growth in C3H/HeJ mice. (a) Representative images of tumor-inclusive Matrigel implants retrieved on day 16 (a scale in mm shown in the background). (b) Image of only Matrigel implant retrieved on day 16. (c) Mean weights of tumors were reduced significantly in mice treated with indomethacin, celecoxib or EP4 antagonist ONO- AE3-208 (EP4A), but not EP1 antagonist ONO-8713 (EP1A), compared with respective vehicle-treated controls. This reduction was significant day 12 with celecoxib and EP4A, and highly significant on day 16 for all therapies except EP1A. Data represent mean (n ¼ 16 per group per day)±s.e. *Po0.05, **Po0.005.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 3 Therapy with non-selective or selective COX-2 inhibitors or EP4 antagonist reduced tumor-associated angiogenesis and lymphangiogenesis in C3H/HeJ mice. (a) Bright field images of Masson’s Tricrome-stained sections (first and third columns) showing peritumoral stroma inclusive of vessels (scale bars in mm given as insets), and fluorescence images of corresponding tumors representing ‘hot spots’ within the tumors (second and fourth columns). Tumor-associated angiogenesis (CD31 immuno-staining in red) and lymphangiogenesis (LYVE-1 immuno-staining in green), are shown as representative fluorescent merged images. There was practically no overlap between the vessels identified by two colors confirming the specificity of the markers. Therapy with COX-1/2 and COX-2 inhibitors or EP4 antagonist but not EP1 antagonist significantly reduced both angiogenesis and lymphangiogenesis. Representative images are shown in (a) and quantified as corresponding ‘hot spot’ scores for CD31 and LYVE-1 (b) (n ¼ 16, using the mean of three hot spots from each of the 16 tumors per group) ±s.e., *Po0.05, **Po0.005.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Staining, Fluorescence, Immunostaining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 4 Therapy with COX-1/2 inhibitor or COX-2 inhibitor or EP4 antagonist abrogated regional and distant lymph node metastasis. Histological pictures (H&E stained) of representative lymph nodes: Day 8: inguinal nodes in control (vehicle treated) (a, inset magnified fourfold in (b) and EP4A treated (c, inset magnified fourfold in d) mice. Lymph node in (a) is completely replaced by tumor cells (marked as T) showing the trail of invasion from the primary tumor on the left. Lymph node in (c) is tumor free. Day 12: nodes in (e, f) show metastatic tumor cells (T), outlined by red markings; lymph nodes in (g, h) are tumor-free. Day 16: nodes in (i, j) show tumor cells (T), outlined by red markings. Nodes in (k, l) are tumor free. Scale bars for magnification in images are shown as insets (50 mm for all, except for a and c, 200 mm).

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Staining, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 5 Therapy with COX-1/2 and COX-2 inhibitors or EP4 antagonist reduces metastatic lung colony formation. (a) Representative images of micro metastases in H&E-stained lung sections on day 16. They were noted as early as day 8 (data not presented). Scale bar represents 100 mm. (b) Median numbers of metastatic lung colonies were reduced with all treatments (except EP1A) at all-time points after tumor transplantation. (n ¼ 8 per group per day). Data represent median±quartile deviations. *Po0.05; **Po0.01.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Staining, Transplantation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 6 Therapy with COX-1/2 inhibitor, COX-2 inhibitor and EP4 antagonist reduces the levels of VEGF-C, -D and phosphorylated AkT proteins in residual tumors. Total tumor lysate proteins (pooled from eight tumors per group, triplicate measurements) were subjected to western blots for pAkT and total AkT, VEGF-C and -D and GAPDH proteins. Densitometric measurements of pAkT relative to total AkT, and VEGF-C and -D relative to GAPDH reveal a significant reduction of all these parameters in the drug-treated groups at all intervals, as compared with vehicle-treated controls. Data represent mean±s.e., *Po0.05.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Targeting COX-2 and EP4 to control tumor growth, angiogenesis, lymphangiogenesis and metastasis to the lungs and lymph nodes in a breast cancer model.

doi: 10.1038/labinvest.2012.90

Figure Lengend Snippet: Figure 7 EP4 antagonist therapy increases the apoptotic/proliferative cell ratios in residual tumors. (a) Representative abundance of proliferative cells labeled for Ki67 marker (red), and apoptotic cells labeled for TUNEL (green) in situ are illustrated in serial sections for day 12 in vehicle-treated (control) and EP4A-treated mice. Images of corresponding H&E-stained tumors in adjacent sections are shown on the left (scale bars are 50 mm, shown as insets). (b) The ratios of apoptotic/proliferative cells at different days following vehicle or EP4A therapy. Data represent mean (n ¼ 12)±s.e., *Po0.05.; **Po0.005 comparing the two groups. The ratios showed a significant increase with time in EP4A-treated mice and decrease in control mice.

Article Snippet: EP4 siRNA products consisted of pools of three to five targetspecific 19–25 nucleotide siRNAs designed to knockdown mouse EP4 gene expression (SC-40174, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and silence select negative control siRNA (Cat. No: 4390843, Ambion) was used as EP4SC siRNA.

Techniques: Labeling, Marker, TUNEL Assay, In Situ, Control, Staining