Journal: Journal of Pathology and Translational Medicine

Article Title: Characteristic Changes in Decidual Gene Expression Signature in Spontaneous Term Parturition

doi: 10.4132/jptm.2016.12.20

Figure Lengend Snippet: TaqMan assays used for qRT-PCR expression profiling

Article Snippet: Prostaglandin signaling molecules , PTGER2 , Prostaglandin E receptor 2 , Hs00168754_m1.

Techniques: Expressing, Membrane, Binding Assay

Journal: Applied Sciences

Article Title: Processed Scutellaria baicalensis Georgi Extract Alleviates LPS-Induced Inflammatory and Oxidative Stress through a Crosstalk between NF-κB and KEAP1/NRF2 Signaling in Macrophage Cells

doi: 10.3390/app11136055

Figure Lengend Snippet: Figure 2. Cell viability and effects of PSGE against LPS-induced NO and PGE2 production and related iNOS, COX-2, mPGES-1 and 15-PGDH expression in RAW 264.7 cells. (A) Cell viability of PSGE in RAW 264.7 cells. RAW 264.7 cells were treated with 50−1000 µg/mL of PSGE for 24 h and cell viability was determined by MTT assay. (B) NO levels were measured by Griess assay, (C) PGE2 levels were measured by ELISA assay, (D) Protein expression and quantitative analysis of (E) iNOS, (F) COX-2, (G) mPGES-1 and (H) 15-PGDH expression relative to β-actin. RAW 264.7 cells were pre-treated with indicated concentration of PSGE or DXM for 2 h followed by LPS for 24 h. DXM was used as positive control. Data shown represent mean ± SEM (n = 3). (### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. LPS).

Article Snippet: RAW 264.7 cells (1 × 105 cells/well) were pretreated with different concentrations of PSGE and DXM 20 μM for 2 h and then exposed to LPS 1μg/mL for 24 h. The supernatant of each well was collected and centrifuged at 1500 rpm at 4 ◦C for 10 min. IL-6 and TNF-α concentrations were measured using mouse IL-6 and TNF-α ELISA kit (Invitrogen, Carlsbad, CA, USA) and PGE2 using mouse PGE2 ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

Techniques: Expressing, MTT Assay, Griess Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Positive Control, Control

Journal: Applied Sciences

Article Title: Processed Scutellaria baicalensis Georgi Extract Alleviates LPS-Induced Inflammatory and Oxidative Stress through a Crosstalk between NF-κB and KEAP1/NRF2 Signaling in Macrophage Cells

doi: 10.3390/app11136055

Figure Lengend Snippet: Figure 3. Effects of PSGE against LPS-induced IL-6, TNF-α cytokine expression, and NF-κB signaling in RAW 294.7 cells. Production of (A) IL-6 and (B) TNF-α levels were measured by ELISA. RAW 264.7 cells were pre-treated with indicated concentration of PSGE or DXM for 2 h followed by LPS for 24 h. (C) Western blot analysis of nuclear and cytosolic fractions using NF-κB p65 antibody, (D) quantitative analysis of nuclear NF-κB p65 relative to Lamin A/C, (E) Protein expression and quantitative analysis of (F) p-IκBα relative to IκBα and (G) HO-1 relative to β-actin. RAW 264.7 cells were pretreated with the indicated concentrations of PSGE and DXM for 2 h followed by LPS for 2 h. Lamin A/C and HSP90 were used as cytosolic and nuclear markers. DXM as positive control. Data shown represent mean ± SEM (n = 3). (### p < 0.001 vs. control; * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. LPS).

Article Snippet: RAW 264.7 cells (1 × 105 cells/well) were pretreated with different concentrations of PSGE and DXM 20 μM for 2 h and then exposed to LPS 1μg/mL for 24 h. The supernatant of each well was collected and centrifuged at 1500 rpm at 4 ◦C for 10 min. IL-6 and TNF-α concentrations were measured using mouse IL-6 and TNF-α ELISA kit (Invitrogen, Carlsbad, CA, USA) and PGE2 using mouse PGE2 ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions.

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

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