Journal: ACS chemical neuroscience

Article Title: Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

doi: 10.1021/acschemneuro.7b00219

Figure Lengend Snippet: NPFF EC50 in functional calcium mobilization assays. (A) NPFF response in stable NPFF1-RD-HGA16 cells (●) and parental RD-HGA16 CHO cells (○). (B) NPFF response in stable NPFF2-RD-HGA16 cells (■) and parental RD-HGA16 CHO cells (○). Representative data from one experiment is shown and each data point is the mean ± SD of duplicate determinations.

Article Snippet: Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from PerkinElmer and used with the Lance Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates.

Techniques: Functional Assay

Journal: ACS chemical neuroscience

Article Title: Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

doi: 10.1021/acschemneuro.7b00219

Figure Lengend Snippet: Antagonist activity of compound 16 in NPFF1 (A) and NPFF2 (B) calcium mobilization functional Ke assays. (A) Concentration-response curves of NPFF alone (○) and NPFF + 5 μM final 16 (□) in stable NPFF1-RD-HGA16 cells. (B) Concentration-response curves of NPFF alone (○) and NPFF + 10 μM final 16 (□) in stable NPFF2-RD-HGA16 cells. The right shift of the NPFF curve in the presence of test compound was used to calculate Ke values as described in the Methods. Representative data from one experiment are shown and each data point is mean ± SD of duplicate determinations.

Article Snippet: Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from PerkinElmer and used with the Lance Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates.

Techniques: Activity Assay, Functional Assay, Concentration Assay

Journal: ACS chemical neuroscience

Article Title: Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists

doi: 10.1021/acschemneuro.7b00219

Figure Lengend Snippet: Antagonist activity of compounds 16 and 33 in NPFF1 (A) and FF2 (B) cAMP functional Ke assays. (A) Concentration-response curves of NPFF alone (○), NPFF + 4 μM final 16 (□), and NPFF + 2 μM final 33 (◊) in stable NPFF1-CHO cells. (B) Concentration-response curves of NPFF alone (○), NPFF + 10 μM final 16 (□), and NPFF + 10 μM final 33 (◊) in stable NPFF2-CHO cells. The right shift of the NPFF curve in the presence of test compound was used to calculcate Ke values as described in the Methods. Each data point is mean ± SEM of at least N=3 conducted in duplicate.

Article Snippet: Stable human NPFFR1-CHO (ES-491-C) and NPFFR2-CHO (ES-490-C) cell lines were purchased from PerkinElmer and used with the Lance Ultra kit (TRF0262) to detect cAMP accumulation in low volume 96-well plates.

Techniques: Activity Assay, Functional Assay, Concentration Assay

Journal: Biomolecules

Article Title: 1-(Arylsulfonyl-isoindol-2-yl)piperazines as 5-HT 6 R Antagonists: Mechanochemical Synthesis, In Vitro Pharmacological Properties and Glioprotective Activity

doi: 10.3390/biom13010012

Figure Lengend Snippet: ( A ): Functional dose-response curve of inhibition of cAMP production at 5-HT 6 R for selected compounds 3e , 3f , and 3g in 1321N1 cells. Data were obtained from three independent experiments run in triplicate. ( B ): Impact of compounds 3e , 3f , and 3g and SB-258585 on basal cAMP production in NG108-15 cells transiently expressing 5-HT 6 R. For each compound, six independent transfection experiments were performed, and data were measured in triplicate. Data are given as means ± SEM of the values.

Article Snippet: The ability of compounds 3e , 3f , and 3g to inhibit 5-CT-induced production of cAMP was assessed using 1321N1 cells expressing the human 5-HT 6 R (PerkinElmer) using previously described procedures [ , ].

Techniques: Functional Assay, Inhibition, Expressing, Transfection

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: CX3CR1 and HIF-1α expression by OvCa tissues: Ovarian tissues from normal and various cancer stages [well-differentiated (Stage I), moderately differentiated (Stage II), and poorly differentiated (Stage III)] were stained with anti-CX3CR1 and anti-HIF-1α antibodies. Magenta (AP) color shows CX3CR1, and Brown (DAB) color shows HIF-1α staining. An Aperio ScanScope CS system with a 40X objective captured digital images of each tissue. Representative cases are immuno-intensities of CX3CR1 and HIF-1α using image analysis Aperio ImageScope v.6.25 software

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Expressing, Staining, Software

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Expression of CX3CR1 and CX3CL1 by OvCa cells. OVCAR-3, SW 626, and TOV-112D cells were stained with FITC-conjugated or anti-CX3CR1 and PE-conjugated isotype or anti-CX3CL1 antibodies, and the expressions were quantified by flow cytometry. The gray and white histograms represent isotypes and CX3CR1/CX3CL1 fluorescence intensity, respectively

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Western blot expression of hypoxia regulatory markers in OvCa cells. Immunoblot detection of OVCAR-3, SW 626, and TOV-112D cells exposed to 3, 6, 9, or 12 h hypoxia respectively. Immunoblotting was accomplished with primary antibodies against HIF-1α, CX3CR1, EMT marker (Snail), and MMP-9. As an internal standard for equal loading, anti-GAPDH antibody was used to probe blots

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Western Blot, Expressing, Marker

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: mRNA analysis of hypoxia-induced signaling molecules in OvCa cells. Ovarian tumor cells (OVCAR-3, SW 626, and TOV-112D) were exposed to hypoxia for 3, 6, 9, or 12 h. Quantitative RT-PCR results for expressions of CX3CR1, HIF-1α, MMP9, Snail, Twist, N- and P-cadherin are shown. The data were normalized to the levels of the housekeeping gene (18 S), and the analyses were accomplished in triplicate. Data were presented as fold change in expression (± standard error); the asterisks indicate significant differences as determined by Student’s t -test (* P < 0.05; ** P < 0.01)

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Migration and invasion induced by the CX3CR1/CX3CL1 interaction in hypoxic OvCa cells. OVCAR-3, SW 626, and TOV-112D cells were cultured in the presence of CoCl 2 (150 μM) (to mimic hypoxia), and with or without CX3CL1 (chemoattractant) and KC7F2 (inhibitor) in the medium. Over 6 days, migration and invasion were assessed. ( a ) Morphology and ( b ) quantitative analysis of surface area, representing the invading cells projecting out of the spheroid into the medium when using CX3CL1. For cells under hypoxia, there was minimal growth, or they remained as aggregates when treated with or without the inhibitor, KC7F2

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Migration, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 1. Identification and charac- terization of GPCR135 ligand activity in rat brain extracts. A, GPCR135 li- gand activity in different rat tissues. Ex- tracted rat tissues at the dilutions shown were used as ligands in the GTPS bind- ing assay using human GPCR135 ex- pressing cell membrane. B, molecular weight characterization of GPCR135 li- gand from rat brain extract. Crude rat brain ethanol/HCl extract was run through a HPLC gel filtration column. Fractions were collected and assayed for GPCR135 ligand activity in GTPS bind- ing assays using human GPCR135 ex- pressing cell membranes. In a parallel ex- periment, peptides and nucleotides with known molecular weights were run using the same conditions to serve as the molec- ular mass standards.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Activity Assay, Membrane, Molecular Weight, Filtration

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 2. Purification and characterization of porcine GPCR135 ligand. After extraction, fractionation, two rounds of ion exchange, and two rounds of reverse phase chromatographies, homogeneous peptide was obtained. A, the final RP-HPLC. Fractions 39–40 showed ligand activity for GPCR135 as demonstrated in GTPS binding assays. B, GTPS binding assays for GPCR135 ligand activity in the RP-HPLC fractions. C, the porcine GPCR135 ligand in fraction 39-40 was subjected to amino acid sequence analysis. The resulting amino acid sequences are aligned to human, mouse, and rat relaxin-3. All cysteine residues are highlighted in bold letters. The three amino acids in porcine GPCR135 ligand that differ from human relaxin-3 are underlined.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Purification, Extraction, Fractionation, Activity Assay, Binding Assay, Sequencing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 3. Expression, purification, and characterization of recombinant human relaxin-3. A, schematic diagram of the predicted amino acid sequence for the human relaxin-3 expression construct, relaxin-3RR. Arrows indicate an enterokinase cleavage site (DDDDK) and two furin cleavage sites (RWRR and RGRR). The amino acid R with the star (*) indicates the mutation made from the natural sequence of RGSR to RGRR, an artificial furin site in the construct. The numbers indicate the corresponding positions of the amino acid in the natural human relaxin-3 pre-propeptide. B, RP-HPLC analysis of purified recombinant human relaxin-3. The purified human relaxin-3 was run in a C-18 RP-HPLC analytical column in 0.1% trifluoroacetic acid with an acetonitrile (ACN) gradient. C, fractions from B were assayed for GPCR135 ligand activity in GTPS binding assays using GPCR135 expressing cell membranes.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Expressing, Purification, Recombinant, Sequencing, Construct, Mutagenesis, Activity Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 4. Characterization of GPCR135 using 125I-relaxin-3 as the radioligand. A, saturation isotherm binding for 125I-relaxin-3 and GPCR135. GPCR135 expressing cell membranes were used in binding assays with different concentrations of 125I-relaxin-3 as the radioligand. Nonspecific binding was determined using the same conditions in the presence of 5 M unlabeled relaxin-3. B, competition binding analysis of GPCR135. GPCR135 expressing cell membranes were used to perform radioligand binding assays with 100 pM 125I-relaxin-3 as the radioligand. Different unlabeled peptides at various concentrations were used as competitors.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Binding Assay, Expressing

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 5. Functional characterization of GPCR135 using relaxin-3 and related peptides as ligands. A, relaxin-3 stimulates 35S-GTPS binding in GPCR135 expressing cells. Different peptides were added at various concentrations to the human GPCR135 expressing cell membranes to stimulate GTPS incorporation. The specific 35S-GTPS incorporation was obtained by subtracting counts without ligand from the counts with ligand. B, inhibition of forskolin-stimulated cAMP production by relaxin-3. Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences). C, dose response of relaxin-3 inhibition of cAMP production in GPCR135 expressing cells. Chinese hamster ovary cells stably expressing GPCR135 were stimulated with different concentrations of peptides, including relaxin-3, at various concentrations. Forskolin was then added to all samples at a final concentration of 5 M. cAMP from the stimulated cells was extracted and measured using cAMP flash plates. D, relaxin-3 stimulates Ca2 mobilization in HEK293 cells co-expressing GPCR135 and Gqi5. HEK293 cells, either mock transfected (293), transfected with Gqi5 (293/Gqi5), human GPCR135 (GPCR135), or co-transfected with human GPCR135 and Gqi5 (GPCR135/Gqi5), were used for Ca2 mobilization assays. Relaxin-3 stimulated intracellular Ca2 mobilization was monitored by FLIPR.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Functional Assay, Binding Assay, Expressing, Inhibition, Stable Transfection, Control, Concentration Assay, Transfection

Journal: Journal of Biological Chemistry

Article Title: Identification of Relaxin-3/INSL7 as an Endogenous Ligand for the Orphan G-protein-coupled Receptor GPCR135

doi: 10.1074/jbc.m308995200

Figure Lengend Snippet: FIG. 6. A, RT-PCR detection of GPCR135 and relaxin-3 mRNA expression profiles in different human tissues. The PCR products were run in 2% agarose gels, stained with ethidium bromide, and visualized under UV irradiation. In parallel, PCRs amplifying human -actin cDNA served as the controls. B–E, coronal brain sections showing expression of GPCR135 and relaxin-3 mRNA by in situ hybridization. B, autoradiogram shows that GPCR135 mRNA distribution is distinct in the paraventricular nucleus (PVN) and supraoptic nucleus (SON). C, brightfield photomicrograph of the paraventricular nucleus showing expression of GPCR135 mRNA. Strong hybridization signal is seen as dark grains. Magnification 20. D, relaxin-3 mRNA distribution in the central gray and nucleus incertus. E, brightfield photomicrograph of central gray (CG) and nucleus incertus (NI) showing expression of relaxin-3 mRNA. Magnification 2. B and D in the figure are pseudocolor images from autoradiograms processed on the Fuji Film Bio-Imaging Analyzer System. Colors represent relative levels of hybridization densities with the rank order of red yellow green blue black.

Article Snippet: Chinese hamster ovary cells stably expressing GPCR135 and control Chinese hamster ovary cells were stimulated with buffer, 200 nM relaxin-3, 5 M forskolin, or 5 M forskolin plus 200 nM relaxin-3. cAMP from the treated cells was extracted and measured using cAMP flash plates (PerkinElmer Life Sciences).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Irradiation, In Situ Hybridization, Hybridization, Imaging

Journal: Animal Cells and Systems

Article Title: Multifunctional antistress effects of standardized aqueous extracts from Hippophae rhamnoides L.

doi: 10.1080/19768354.2016.1250816

Figure Lengend Snippet: Figure 3. The effects of HR extracts on 5-HT-medicated cAMP formation in human astrocytoma 1321n1 cells, stably expressed with the human serotonin 5-HT6 receptor gene. Test extracts were treated in the presence of 100 µM 5-HT, a receptor agonist. After 15 min of treatment with the extracts, intracellular cAMP levels were measured using a ParameterTM cAMP Assay according to the manufacturer’s instructions. The data express the mean latency ± SD. Different letters above the bar indicate significant differences, as determined by ANOVA (p < .05, upper case: compared in 10 μg/mL conc., lower case: compared in 5 μg/mL conc.).

Article Snippet: All imaging data were collected and analyzed using Universal Imaging Software (West Chester, PA) (Kim et al. 2004). cAMP accumulation assay through 5-HT6 receptor Human astrocytoma 1321N1 cells that stably expressed the human serotonin 5-HT6 receptor gene (cat. ES-316CV, clone C1, Perkin-Elmer, Boston, MA, USA) were grown in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 0.4 mg/mL Geneticin (Calbiochem, Darmstadt, Germany) for receptor expression selection at 37°C in a 5% CO2 incubator.

Techniques: Stable Transfection, cAMP Assay