Journal: bioRxiv

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting prostaglandin E2 signaling

doi: 10.1101/2023.04.04.535590

Figure Lengend Snippet: A-B , NeuN immunofluorescence (A) and quantification (B) showing enhanced neurodegeneration in PGE2 treated compared to DMSO treated and PGD2 treated COCS after prion infection. n = 18 slices/condition. C-D , NeuN immunofluorescence (C) and quantification (D) showing diminished neurodegeneration in COCS treated with Ptges inhibitors C118 and C934 compared to DMSO treated COCS after prion infection. n = 18 slices/condition. E , Survival curves showing decelerated prion disease in Cox2 knockout (Cox2Luc) mice compared to littermate WT mice. F-G , Map2 immunofluorescence (F) and quantification (G) showing diminished dendritic pathology in the hippocampi of Cox2Luc mice after prion inoculation compared to littermate WT mice. Brain samples were collected at 21 wpi. n = 6 mice/group.

Article Snippet: Lenti-vectors used for expressing human PGE2 receptors EP1 (pLenti-PTGER1-mGFP-P2A- Puro, RC208597L4), EP2 (pLenti-PTGER2-mGFP-P2A-Puro, RC210883L4), EP3 (pLenti- PTGER3-mGFP-P2A-Puro, RC220173L4) and EP4 (pLenti-PTGER4-mGFP-P2A-Puro, RC210932L4) were purchased from OriGene Technologies.

Techniques: Immunofluorescence, Infection, Knock-Out

Journal: bioRxiv

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting prostaglandin E2 signaling

doi: 10.1101/2023.04.04.535590

Figure Lengend Snippet: A-B , NeuN immunofluorescence (A) and quantification (B) showing that the enhanced neurodegeneration in prion-infected, NG2 glia-depleted (CP673451) COCS can be rescued by treatment with Ptges inhibitors C118 and C934. n = 16 slices/condition. C , Cox2 ablation (Cox2Luc) suppresses the acceleration of prion disease in NG2 glia-depleted (Pdgfrα iDTR ) mice. NG2 glia depletion was induced at 16 wpi. D-E , Map2 immunofluorescence (D) and quantification (E) showing enhanced dendritic pathology in NG2 glia-depleted (Pdgfrα iDTR ) hippocampi of prion-infected mice, and its rescue by Cox2 ablation (Cox2Luc). n = 6 mice/group. F-G , Live-cell images (F) and quantitative analysis (G) of chronically prion infected HovS cells expressing control (Ctrl) transgene or one of the four PGE2 receptors (Ptger). 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; 4 technical repeats/experiment

Article Snippet: Lenti-vectors used for expressing human PGE2 receptors EP1 (pLenti-PTGER1-mGFP-P2A- Puro, RC208597L4), EP2 (pLenti-PTGER2-mGFP-P2A-Puro, RC210883L4), EP3 (pLenti- PTGER3-mGFP-P2A-Puro, RC220173L4) and EP4 (pLenti-PTGER4-mGFP-P2A-Puro, RC210932L4) were purchased from OriGene Technologies.

Techniques: Immunofluorescence, Infection, Expressing, Control

Journal: International journal of cancer

Article Title: Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer.

doi: 10.1002/ijc.28076

Figure Lengend Snippet: Figure 1. Inhibiting EP1 receptor signaling reduces the growth of pre-established tumors in vivo. (a) CT26 colon tumor cells (2 3 105) were subcutaneously inoculated into BALB/c mice. Inoculated tumor cells and treatments are indicated. Unless otherwise indicated, ONO-8713 was administered in the basal diet. Tumor growth was monitored as described in the Material and Methods section. Data points represent the mean value 6 SEM and show the findings of one tumor challenge experiment. (b) EP1 receptor expression was suppressed in CT26EP1 shRNA cells. The efficiency of suppression of EP1 receptor was assessed by RT-PCR and Western blotting. For the RT-PCR, data were normalized to b-actin and analyzed using the 22DDCt method. Immunoblotting for b-actin was used as the loading control. (c) Proliferation of tumor cells ex vivo was unaffected by EP1 receptor expression or EP1 receptor signaling. Cell proliferation was determined by resazurin reduction. Proliferation was normalized to tumor cells isolated from CT26scr shRNA tumors. Data shown are the findings from three independent experiments. (d) Blocking EP1 receptor signaling does not affect PGE2 secretion by tumor cells. Tumor cells were isolated from all tumor-bearing mice and cultured ex vivo for 24 hr. Cell culture supernatants were harvested, and the level of PGE2 was measured in triplicate by ELISA. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Generation of FasLlow/negative and EP1low/negative colon cancer cells Cells were transfected with lentiviral particles containing three target-specific shRNAs against FasL (sc-35358-V), EP1 (sc40170-V) or control lentiviral particles containing scrambled shRNA (sc-108080; Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturers’ instructions.

Techniques: In Vivo, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Ex Vivo, Isolation, Blocking Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International journal of cancer

Article Title: Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer.

doi: 10.1002/ijc.28076

Figure Lengend Snippet: Figure 2. Signaling through the EP1 receptor upregulates FasL expression in vivo. (a) CT26 cells were treated with increasing concentrations of ONO-8713 for 24 hr, or FasL and EP1 receptor expression was suppressed in CT26FasL shRNA and CT26EP1 shRNA cells, respectively. FasL expression was determined by Western blotting. Expression of the housekeeping gene b-actin was used as an internal control. Results shown are represen- tative of three independent experiments. (b) FasL expression was suppressed in CT26FasL shRNA cells. The efficiency of suppression of FasL was assessed by RT-PCR and Western blotting. RT-PCR data were normalized to b-actin and analyzed using the 22DDCt method. (c) Suppression of FasL expression reduces tumor development in vivo. Cells were subcutaneously inoculated into BALB/c mice. Mice were palpated thrice weekly to detect tumors. Data shown are the percentage of mice remaining tumor free on the indicated day. (d) Suppression of FasL expression reduces tumor growth in vivo. CT26scr shRNA or CT26FasL shRNA cells (2 3 105) were subcutaneously inoculated into BALB/c mice. ONO-8713 was adminis- tered in the basal diet with the day of initiation of treatment indicated. Tumor growth was monitored as described in the Material and Methods section. Data points represent the mean value 6 SEM and show the findings of one tumor challenge experiment. (e) Blocking EP1 signaling suppresses FasL expression in vivo. Tumors were excised after 48 days. FasL expression in the excised tumors was analyzed by RT-PCR and Western blotting. For Western blotting, 30 mg of protein from all tumors per treatment group was pooled. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Article Snippet: Generation of FasLlow/negative and EP1low/negative colon cancer cells Cells were transfected with lentiviral particles containing three target-specific shRNAs against FasL (sc-35358-V), EP1 (sc40170-V) or control lentiviral particles containing scrambled shRNA (sc-108080; Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturers’ instructions.

Techniques: Expressing, In Vivo, shRNA, Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Blocking Assay

Journal: International journal of cancer

Article Title: Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer.

doi: 10.1002/ijc.28076

Figure Lengend Snippet: Figure 3. Blocking EP1 receptor signaling alters the intratumoral immune cell population. (a) Flow cytometric analysis revealed that suppressing EP1 receptor signaling significantly reduces the percentage of CD41 T cells and increases the percentage of CD81

Article Snippet: Generation of FasLlow/negative and EP1low/negative colon cancer cells Cells were transfected with lentiviral particles containing three target-specific shRNAs against FasL (sc-35358-V), EP1 (sc40170-V) or control lentiviral particles containing scrambled shRNA (sc-108080; Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturers’ instructions.

Techniques: Blocking Assay

Journal: International journal of cancer

Article Title: Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer.

doi: 10.1002/ijc.28076

Figure Lengend Snippet: Figure 4. Antagonizing the EP1 receptor reduces macrophage infiltration but does not alter macrophage polarization. (a) Flow cytometric analysis revealed that suppressing EP1 receptor signaling reduces the percentage of F4/801 cells within the tumors. Data points represent the mean 6 SEM; n 5 mice per group. (b) The EP1 receptor does not play a role in TAM polarization. Macrophages were isolated from a minimum of five excised tumors per treatment group as described in the Material and Methods section. RNA was isolated and pooled, and the changes in transcription of IL-12, IL-6 and NOS2 were determined by RT-PCR. Data were normalized to b2-microglobulin and analyzed using the 22DDCt method. Results represent the mean 6 SEM. (c) Blocking signaling through the EP1 receptor does not alter secretion of PGE2 by TAM. Isolated macrophages (n 5 3 per group) were cultured ex vivo for 24 hr. Cell culture supernatants were harvested, and the level of PGE2 was measured in triplicate by ELISA.

Article Snippet: Generation of FasLlow/negative and EP1low/negative colon cancer cells Cells were transfected with lentiviral particles containing three target-specific shRNAs against FasL (sc-35358-V), EP1 (sc40170-V) or control lentiviral particles containing scrambled shRNA (sc-108080; Santa Cruz Biotechnology, Santa Cruz, CA), according to the manufacturers’ instructions.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Journal of Bone and Mineral Research

Article Title: PGE2 Signaling Through the EP4 Receptor on Fibroblasts Upregulates RANKL and Stimulates Osteolysis

doi: 10.1359/JBMR.090412

Figure Lengend Snippet: PE-induced osteolysis is independent of EP1 and EP2 signaling. WT, EP1−/−, and EP2−/− mice (n = 8) received in vivo μCT scans before and 10 days after sham or PE surgeries to induce osteolysis. (A) Representative reconstructed 3D images of day 10 calvaria and osteolytic volume from each group are shown. (B) The osteolytic volumes from each group are presented as mean ± SD (*p < 0.05 vs. sham group).

Article Snippet: Briefly, fibroblasts were isolated and plated as described above then transfected with EP1 siRNA (Santa Cruz Biotechnology) according to the manufacturer's instructions.

Techniques: In Vivo

Journal: Journal of Bone and Mineral Research

Article Title: PGE2 Signaling Through the EP4 Receptor on Fibroblasts Upregulates RANKL and Stimulates Osteolysis

doi: 10.1359/JBMR.090412

Figure Lengend Snippet: Particle and PGE2-induced RANKL expression in fibroblasts is independent of EP1 and EP2 signaling in vitro. Fibroblasts were isolated from fractions 1 and 2 of calvaria digestion from WT, EP1−/−, and EP2−/− mice and treated with PBS (control, “C”), Ti (5 × 106 particles/ml) or PGE2 (10−6 M). Total RNA was extracted for real-time PCR after 5 h, and the data are presented as the mean (n = 3) ± SD (A, C, and E). Total protein extracts were obtained from cell lysates after 24 h, and RANKL expression was examined compared with β-actin loading control by Western blot. Representative Western blots from two independent experiments are shown (B, D, and F). siRNA was used to specifically knockdown expression of EP1 in EP2−/− fibroblasts, and cells were examined for RANKL mRNA (G) and protein expression (H). (*p < 0.05 vs. PBS control)

Article Snippet: Briefly, fibroblasts were isolated and plated as described above then transfected with EP1 siRNA (Santa Cruz Biotechnology) according to the manufacturer's instructions.

Techniques: Expressing, In Vitro, Isolation, Control, Real-time Polymerase Chain Reaction, Western Blot, Knockdown

Journal: bioRxiv

Article Title: The nuclear receptor Nr2f6 represses skeletal muscle oxidative metabolism and force production

doi: 10.1101/2023.05.17.540900

Figure Lengend Snippet: (A) Relative gene expression using RT-qPCR in stable Nr2f6-myc overexpression myotubes. N=4-5. (B) Densitometry and representative images of PGC-1α western blot in stable Nr2f6-myc overexpression myotubes. N=5. (C) Relative gene expression using RT-qPCR in stable Nr2f6 knockdown myotubes. N=3-5. (D) Luciferase reporter assay in HEK293 cells with 2 kbp PGC1Α promoter overexpressing HA-tagged Nr2f6 or control empty vector (EV). (E) Luciferase activity of UCP3 promoter transactivation assay in cells overexpressing Nr2f6-myc and siNr2f6 transfected cells. N=3. (F) Mouse UCP3 genomic locus retrieved from UCSC Genome Browser with the Nr2f6 response element highlighted. Top tracks: ChIP-seq of Myogenin and MyoD at 24h and 60h of differentiation. Middle track: DNAse hypersensitivity assay, with open sensitive regions in grey. Bottom tracks: histone marks ChIP-seq. (G) Relative gene expression using RT-qPCR in human primary skeletal myotubes transfected with siNr2f6 or siScr. N=6. Boxplot with whiskers spanning minimum to maximal and box edges 25 th -75 th percentile, the line at the median and + at the mean. * Indicates p < 0.05 using unpaired two-tailed Student’s t-test. The numbers above some bars indicate the p-value.

Article Snippet: Luciferase reporter assays were performed in MEF cells transfected with a UCP3 reporter plasmid ( ) (UCP3 EP1, Addgene #71743) or PGC-1α 2kb promoter ( ) (Addgene #8887), normalization plasmid coding for Renilla luciferase (pRL-SV40) and either control empty vector or Nr2f6 coding plasmid (Gift from Dr. Gottfried Baier, Medical University of Innsbruck, Austria) using Lipofectamine 3000.

Techniques: Gene Expression, Quantitative RT-PCR, Over Expression, Western Blot, Knockdown, Luciferase, Reporter Assay, Control, Plasmid Preparation, Activity Assay, Transactivation Assay, Transfection, ChIP-sequencing, Two Tailed Test