ep3  (Proteintech)

Journal: International Journal of Molecular Sciences

Article Title: Differential Fatty Acid Response of Resident Macrophages in Human Skeletal Muscle Fiber and Intermuscular Adipose Tissue

doi: 10.3390/ijms251910722

Figure Lengend Snippet: Representative images of skeletal muscle tissue samples. All images were obtained from participant P10. ( a ) The image of skeletal muscle tissue after HE staining indicating areas of SMFs and IMATs. ( b ) Immunofluorescence images of 230 µm × 260 µm fields of view after co-staining with DAPI and secondary antibodies as negative controls. ( c ) Immunofluorescence images of 230 µm × 260 µm fields of view after co-staining with primary antibodies against CD80, CD11c, MARCO, CD163, CD206, or PTGER3 with the corresponding secondary antibodies and DAPI. The small panels on the left show magnified single-cell images (dashed line circles) with DAPI (blue, top), IgG594 (red, middle), or IgG488 (green, middle) filters. DAPI and IgG594 or DAPI and IgG488 were merged (Merge, bottom) to determine the specificity of the detected signals. Verified positive cells are labeled with white arrowheads.

Article Snippet: Paraffin tissue slides were deparaffinized and then heated in citrate buffer (pH 6.0) for 30 min. After cooling in water, the slides were washed twice in 0.1% Tween 20 (9127.1, Carl Roth) in PBS and blocked in UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Dallas, TX, USA) for 60 min. Primary antibodies against human CD80 (1:1000, ab134120, Abcam, Cambridge, UK), CD11c (1:100, ab52632, Abcam), MARCO (1:100, PA5-64134, Thermo Fisher Scientific), CD163 (1:200, ab156769, Abcam), CD206 (1:100, PA5-101657, Thermo Fisher Scientific), PTGER3 (1:100, PA5-102057, Thermo Fisher Scientific), or VDAC1 (1:100, ab154856, Abcam) were diluted in 3% BSA in PBS as indicated.

Techniques: Staining, Immunofluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Differential Fatty Acid Response of Resident Macrophages in Human Skeletal Muscle Fiber and Intermuscular Adipose Tissue

doi: 10.3390/ijms251910722

Figure Lengend Snippet: Different sets of cytokines/chemokines regulate the number of PTGER3 + macrophages in adjacent SMFs and IMATs. Pearson correlation and Spearman’s rank correlation analyses were employed to determine the relationships between the relative fold changes in PTGER3 + ( a , b ) or CD206 + ( c ) macrophage numbers in IMATs ( a ) or SMFs ( b , c ) of the donors ( n = 12) and the tissue expression levels of different cytokines/chemokines ( y -axis). The correlation coefficients (r) and significance levels ( p ) for the relationships are presented on the top right or left side of the diagrams.

Article Snippet: Paraffin tissue slides were deparaffinized and then heated in citrate buffer (pH 6.0) for 30 min. After cooling in water, the slides were washed twice in 0.1% Tween 20 (9127.1, Carl Roth) in PBS and blocked in UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Dallas, TX, USA) for 60 min. Primary antibodies against human CD80 (1:1000, ab134120, Abcam, Cambridge, UK), CD11c (1:100, ab52632, Abcam), MARCO (1:100, PA5-64134, Thermo Fisher Scientific), CD163 (1:200, ab156769, Abcam), CD206 (1:100, PA5-101657, Thermo Fisher Scientific), PTGER3 (1:100, PA5-102057, Thermo Fisher Scientific), or VDAC1 (1:100, ab154856, Abcam) were diluted in 3% BSA in PBS as indicated.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Differential Fatty Acid Response of Resident Macrophages in Human Skeletal Muscle Fiber and Intermuscular Adipose Tissue

doi: 10.3390/ijms251910722

Figure Lengend Snippet: Significance levels of FA-mediated accumulation in IMAT-or SMF-resident macrophages.

Article Snippet: Paraffin tissue slides were deparaffinized and then heated in citrate buffer (pH 6.0) for 30 min. After cooling in water, the slides were washed twice in 0.1% Tween 20 (9127.1, Carl Roth) in PBS and blocked in UltraCruz Blocking Reagent (Santa Cruz Biotechnology, Dallas, TX, USA) for 60 min. Primary antibodies against human CD80 (1:1000, ab134120, Abcam, Cambridge, UK), CD11c (1:100, ab52632, Abcam), MARCO (1:100, PA5-64134, Thermo Fisher Scientific), CD163 (1:200, ab156769, Abcam), CD206 (1:100, PA5-101657, Thermo Fisher Scientific), PTGER3 (1:100, PA5-102057, Thermo Fisher Scientific), or VDAC1 (1:100, ab154856, Abcam) were diluted in 3% BSA in PBS as indicated.

Techniques:

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: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Prostaglandin E receptor 3 (PTGER3) knockdown promotes triple‐negative breast cancer (TNBC) cell migration, invasion, and proliferation. (A) Western blotting and quantitative analysis showing that PTGER3 expression was relatively low in MDA‐MB‐231 cells and high in MCF‐7 cells. (B, C) Western blot and quantitative analysis showing the expression of PTGER3 in MDA‐MB‐231 and MCF‐7 cells after transfection with the up‐/downregulated plasmids. (D, E) Wound‐healing assay was performed on MDA‐MB‐231 and MCF‐7 cells with altered PTGER3 expression. Scale bars: 100 μm. (F, G) Migration and invasion assays were performed on MDA‐MB‐231 and MCF‐7 cells with altered PTGER3 expression. Scale bars: 100 μm. (H, I) Colony formation assay was performed on MDA‐MB‐231 and MCF‐7 cells with altered PTGER3 expression. (J, K) MTT assay was performed on MDA‐MB‐231 and MCF‐7 cells with altered PTGER3 expression. Data are presented as the mean ± SD. Student's t ‐test result.

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: Knockdown, Migration, Western Blot, Expressing, Transfection, Wound Healing Assay, Colony Assay, MTT Assay

Journal: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Downregulation of prostaglandin E receptor 3 (PTGER3) enhances epithelial–mesenchymal transition (EMT) in triple‐negative breast cancer (TNBC) cells. (A) Western blotting was used to assess the protein levels of EMT‐related molecules. (B) GSEA analysis showed that low PTGER3 expression was associated with the TGFβ pathway of EMT. (C, D) Analysis of the GSE76275 dataset revealed significant negative correlations between PTGER3 expression and expression of the EMT molecules CDH2 and TFCP2. (E, F) sh2‐PTGER3‐transfected MDA‐MB‐231 cells showed more mesenchymal morphology (red arrow), while PTGER3‐overexpressing MDA‐MB‐231 cells showed more epithelial morphology. (G, H) MDA‐MB‐231 and MCF‐7 cells stably transfected with plasmids were used in the tubule formation assay to determine VM formation ability. Data are presented as the mean ± SD. Student's t ‐test results.

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: Western Blot, Expressing, Transfection, Stable Transfection, Tube Formation Assay

Journal: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Prostaglandin E receptor 3 (PTGER3) affects ferroptosis of triple‐negative breast cancer (TNBC) cells by targeting GPX4. (A) Western blotting showing increased GPX4 expression in PTGER3‐knockdown MDA‐MB‐231 cells and decreased GPX4 expression in PTGER3‐overexpression MDA‐MB‐231 cells. RIPK1 expression showed no significant changes. (B) Subcellular localization of PTGER3 expression. (C) PTGER3 binding site sequence as predicted from JASPAR. (D) The luciferase activity of cells cotransfected with the ex‐PTEGR3 plasmid and GPX4 promoter was significantly lower compared with other groups. (E) With increasing ex‐PTEGR3 plasmid dose, the luciferase activity gradually declined. This dose–response pattern suggests that PTGER3 binds to the GPX4 promoter. (F–M) GSH (F, G), ROS (H, I), MDA (J, K), and ferrous ion (L, M) levels in cells treated with DMSO, erastin, and erastin+Fer‐1 (scale bars: 100 μm). Data are presented as the mean ± SD. One‐way ANOVA.

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Binding Assay, Sequencing, Luciferase, Activity Assay, Plasmid Preparation

Journal: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Prostaglandin E receptor 3 (PTGER3) affects triple‐negative breast cancer (TNBC) cell survival and growth in vivo by affecting ferroptosis. (A) Overexpression of PTGER3 had an inhibitory effect on tumor growth. (B) Hematoxylin–eosin (H&E) staining of the xenografts. Immunohistochemistry (IHC) staining was used to evaluate the expression of PTGER3, Ki67, E‐cadherin, vimentin, RIPK1, and GPX4 (scale bars: 50 μm). (C) GPX4 protein expression in breast cancer tissue samples and corresponding noncancer tissue samples (scale bars: 200 μm). (D) Violin plot showing the relative expression of GPX4 in TNBC and non‐TNBC tissues from the GSE76275 dataset (*** p < 0.001). (E) Kaplan–Meier survival curve showing the prognostic value of GPX4 expression in TNBC patients. (F) Significant negative correlation between PTGER3 expression and GPX4 expression in samples from the GSE76275 dataset.

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: In Vivo, Over Expression, Staining, Immunohistochemistry, Expressing

Journal: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Activation of the PI3K‐AKT pathway in prostaglandin E receptor 3 (PTGER3)‐downregulated triple‐negative breast cancer (TNBC) cells. (A) KEGG pathway enrichment of PTGER3‐related genes identified the PI3K‐AKT pathway as the top‐ranked pathway. (B) GSEA analysis showed that low PTGER3 expression was closely associated with the PI3K‐AKT pathway. (C) Western blotting showed that PTGER3 knockdown promoted AKT phosphorylation in MDA‐MB‐231 cells, whereas PTGER3 overexpression inhibited AKT phosphorylation. (D) Following the addition of MK‐2206 to MDA‐MB‐231 cells with altered PTGER3 expression, the expression of p‐AKT decreased, but GPX4 expression did not change significantly. (E, F) Evaluation of GSH content in cells after different treatments. (G, H) Quantitative analysis using the DHE probe to determine reactive oxygen species (ROS) levels in cells after different treatments (erastin: 2 μM; MK‐2206: 5 μM; Fer‐1: 5 μM) Scale bars: 100 μm. Data are presented as the mean ± SD. One‐way ANOVA.

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: Activation Assay, Expressing, Western Blot, Knockdown, Phospho-proteomics, Over Expression

Journal: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Triple‐negative breast cancer (TNBC) cells with prostaglandin E receptor 3 (PTGER3) overexpression showed increased sensitivity to paclitaxel (PTX) and inhibition of cell proliferation. (A–D) MTT assay was used to determine the effect of PTX and PTX + erastin on the proliferation of MDA‐B‐231 cells (A, B) and MCF‐7 cells (C, D) with altered PTGER3 expression. (E, F) Colony formation assay was used to evaluate the effect of PTX and PTX + erastin on the colony‐forming ability of MDA‐MB‐231 cells (E) and MCF‐7 cells (F) with altered PTGER3 expression (erastin: 2 μM; PTX: 0.25 μM; Fer‐1: 5 μM). Data are presented as the mean ± SD. One‐way ANOVA.

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: Over Expression, Inhibition, MTT Assay, Expressing, Colony Assay

Journal: Cancer Science

Article Title: PTGER3 knockdown inhibits the vulnerability of triple‐negative breast cancer to ferroptosis

doi: 10.1111/cas.16169

Figure Lengend Snippet: Schematic of the proposed mechanism by which the PTGER3‐GPX4/GSH‐PI3K‐AKT axis affects ferroptosis in triple‐negative breast cancer (TNBC) (by Figdraw).

Article Snippet: The following primary antibodies were used: PTGER3 (1:900 dilution, MAB102431‐SP) from Novus, Vimentin (1:1000, ab92547) from Abcam, phospho‐AKT (S473) (1:500, T40067F) from Abmart, GPX4 (1:1000, #DF6701) and AKT (1:500, #AF6259) from Affinity, E‐cadherin (1:1000, #3195) from Cell Signaling Technology, RIPK1 (1:900, 17519‐1‐AP), and MMP‐2 (1:500, 10373‐2‐AP) from Proteintech.

Techniques: