Journal: The Journal of Clinical Investigation

Article Title: Maresin 1 activates LGR6 receptor promoting phagocyte immunoresolvent functions

doi: 10.1172/JCI129448

Figure Lengend Snippet: (A) A panel of orphan GPCRs was screened in the presence of 10-nM MaR1 or vehicle (0.1% ethanol) using the β-arrestin PathHunter GPCR system. The % activity = 100% × (mean RLU of test sample – mean RLU of vehicle control)/(mean RLU of vehicle control). (B–E) Ligand (MaR1)-receptor interaction was monitored using the CHO-β-arrestin system overexpressing LGR6 or GPR148. Results are mean ± SEM from 3 independent experiments. (B) LGR6 or GPR148 cells with MaR1. #P < 0.05, ##P < 0.01. MaR1 versus vehicle with LGR6 cells. *P < 0.05, ***P < 0.001. LGR6 versus GPR148. (C) LGR6 cells with MaR1 or MaR1 ME. *P < 0.05, **P < 0.01; ***P < 0.001 versus vehicle. (D) LGR6 cells with MaR1, MCTR1, MCTR2, or MCTR3. *P < 0.05, **P < 0.01; ***P < 0.001 versus vehicle. #P < 0.05, ##P < 0.01; ###P < 0.001 versus MaR1. (E) LGR6 cells with MaR1, Rspo-2, or Rspo-2+MaR1. *P < 0.05, **P < 0.01; ***P < 0.001 versus vehicle. #P < 0.05, ##P < 0.01; ###P < 0.001 versus MaR1. For D and E, the 6 groups (MaR1, MCTR1, MCTR2, MCTR3, Rspo-2, Rspo-2+MaR1) were carried out in the same experiments (n = 3). For clarity, the results were separated into D and E. The same MaR1 response curve is presented in both panels for direct comparisons. The statistical analysis (2-way ANOVA with Tukey’s multiple comparisons test) was carried out with all 6 groups. (F) MaR1 (0.1–10 nM) was incubated with CHO-β-arrestin-LGR6 at 4°C, 25°C, 37°C, or 40°C. Results are mean ± SEM from 3 independent experiments. #P < 0.05, versus 4°C; **P < 0.01, versus 4°C and 25°C. (G) Intracellular cAMP. HEK cells transfected with human LGR6 or mock plasmids were incubated with 1- to 100-nM MaR1 for 15 minutes, and cAMP levels were determined. Results are mean ± SEM from 4 independent experiments. ***P < 0.001, versus HEK-mock cells; ###P < 0.001 versus vehicle control. (B–G) Statistical analysis was carried out using 2-way ANOVA with Tukey’s multiple comparisons test. (H) Maresin biosynthesis pathways.

Article Snippet: Test compounds were incubated with cells for 1 hour at 37°C, and receptor activation was determined by measuring chemiluminescence using the PathHunter detection kit (DiscoverX). cAMP measurements.

Techniques: Activity Assay, Incubation, Transfection

Journal: The Journal of Cell Biology

Article Title: Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis

doi: 10.1083/jcb.202207097

Figure Lengend Snippet: Senescent cells exhibit increased CD47 expression in vivo and in vitro. (A) Violin plots showing Cd47, SIRPα and Cdkn1a expression in lung tissue as assayed by the Tabula Muris Senis dataset. *P < 0.05, ****P < 0.0001. Statistically significant differences were determined by Wilcoxon test. Expression levels obtained from RNA-sequencing data for liver fibrosis rat models . (B and C) Transcripts per million (tpm) expression values for Cd47 , Sirpα , and Cdkn1a in rat livers derived frombile duct ligation (BDL; B) or thioacetamide (TAA)-induced liver fibrosis (C). Data are represented as mean ± SEM. Significant de-regulation (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001) was determined by limma/voom based on counts derived from featureCounts. (D) Representative immunofluorescence images showing that p21 staining (red) co-localized with enhanced CD47 expression (green) in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in mice upon feeding with a high fat diet. Scale bar: 50 µm. n = 3–4 mice per group. (E) Quantification of p21-positive cells per field and the percentual area of CD47 staining in SAT and VAT from animals fed a high fat diet (HFD) compared to normal diet (ND). All values are means ± SEM; *P < 0.05. Statistically significant differences were determined by t test or Mann-Whitney test, as appropriate; n = 3–4 mice per group.

Article Snippet: The next day SIRPα binding was quantified by employing an SIRPα PathHunter Bioassay Detection Kit (93-0933; Eurofins), which was co-developed with Eurofins.

Techniques: Expressing, In Vivo, In Vitro, RNA Sequencing, Derivative Assay, Ligation, Immunofluorescence, Staining, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: Senescent cells suppress macrophage-mediated corpse removal via upregulation of the CD47-QPCT/L axis

doi: 10.1083/jcb.202207097

Figure Lengend Snippet: QPCT/L is essential for mediating the suppression of efferocytosis through the CD47-SIRPα axis. (A and B) Proteomic analysis of proliferating versus senescent NHLF (A) and Panc1 cells (B). Volcano plot representing the negative log10-transformed P values vs. the log 2 fold changes in protein intensities of senescent compared to proliferating cells. Dotted lines depict a P value of 0.05 and a fold change of 1.5. Senescence was induced by chemical treatment. Relative QPCTL (red symbol) and CD47 (blue symbol) protein expression normalized to the proliferating control. n = 3 biological replicates. (C) Quantification the relative QPCTL enzyme activity. Enzyme activity of senescent cells was normalized to the respective proliferating control (white bar). Data are representative of two independent experiments. All values are means ± SEM. (D) SIRPα binding of proliferating (white bar) or senescent cells (orange bar) was quantified using a luminescent reporter assay. All values are means ± SEM. Statistically significant differences were determined by unpaired Student’s t test; n = 3–6 biological replicates. (E) Schematic overview of the overall experimental design. Senescent fibroblasts were incubated with neutralizing anti-SIRPα antibodies (mouse) or FAB fragments (human) for 1 h, then direct co-cultures with primary macrophages were assembled for 6 h, and then exposed to labeled apoptotic corpses. Corpse removal was monitored by flow cytometry (mouse) or live cell imaging (human). (F) Efferocytosis in the presence of senescent 3T3 cells (induced by palbociclib) and a SIRPα blocking antibody. Data are representative of three independent experiments. All values are means ± SEM. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction; ****P < 0.0001. n = 3 biological replicates. (G) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and senescent primary IPF-derived fibroblast (IPF). Efferocytotic capability of macrophages in co-culture with senescent fibroblast in the presence (magenta line) or absence of FAB fragments (black line) was monitored over time using the IncuCyte S3 system. Data are representative of three independent experiments. All values are means ± SEM. (H) QPCTL activity was quantified in proliferating or senescent primary IPF-derived lung fibroblasts in the presence (5, 10 µM) or absence of the QPCTL inhibitor SEN-177. Enzyme activity of senescent cells was normalized to the respective proliferating control (gray bar). Data are representative of four independent experiments. All values are means ± SEM. **P < 0.005. Statistically significant differences were determined by one-way ANOVA with Tukey correction. (I) SIRPα binding of proliferating or senescent primary IPF-derived lung fibroblasts in the presence (1, 5, 10 µM) or absence of the QPCTL inhibitor SEN-177 was quantified using a luminescent reporter assay. Data are representative of three independent experiments. All values are means ± SEM. ****P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction. (J) Quantification of efferocytosis of apoptotic corpses in co-cultures of human MDMs and proliferating (gray bar) or senescent IPF-derived lung fibroblasts. Senescent IPF-derived lung fibroblasts were treated with the QPCTL inhibitor SEN-177 (1, 5, 10 µM) prior the assembly of the co-culture with MDMs. Efferocytotic capability of macrophages was monitored over time using the IncuCyte S3 system. Then area under curve (AUC) from 2 to 22 h was calculated and plotted. Data are representative of three independent experiments. All values are means ± SEM. *P < 0.05, **P < 0.005. Statistically significant differences were determined by one-way ANOVA with Bonferroni correction.

Article Snippet: The next day SIRPα binding was quantified by employing an SIRPα PathHunter Bioassay Detection Kit (93-0933; Eurofins), which was co-developed with Eurofins.

Techniques: Transformation Assay, Expressing, Control, Activity Assay, Binding Assay, Reporter Assay, Incubation, Labeling, Flow Cytometry, Live Cell Imaging, Blocking Assay, Derivative Assay, Co-Culture Assay

Journal: Heliyon

Article Title: Novel inverse agonists for the orphan G protein-coupled receptor 6

doi: 10.1016/j.heliyon.2018.e00933

Figure Lengend Snippet: Constitutive activity of GPR6. (A) Constitutive activity of GPR6 in the cAMP accumulation assays. The open bar represents parental HEK293 cells, while the striped bar represents HEK293 cells stably expressing GPR6. *Significant difference from parental HEK293 cells ( p < 0.05, t test). (B) Constitutive activity of GPR6 in the β-arrestin2 recruitment assays. The open bar represents CHO parental cells expressing the EA-β-arrestin2, while the striped bar represents CHO cells co-expressing both GPR6-PK1 and EA-β-arrestin2. *Significant difference from parental cells ( p < 0.05, t test).

Article Snippet: The PathHunter TM Chinese hamster ovary (CHO)-K1 β-arrestin2 human GPR6 eXpress kits were purchased from DiscoverX (Fremont, CA).

Techniques: Activity Assay, Stable Transfection, Expressing

Journal: Heliyon

Article Title: Novel inverse agonists for the orphan G protein-coupled receptor 6

doi: 10.1016/j.heliyon.2018.e00933

Figure Lengend Snippet: Effects of endogenous and phyto-cannabinoids on GPR6-mediated cAMP accumulation and β-arrestin2 recruitment. (A) Effects of endocannabinoids on cAMP accumulation. (B) Effects of endocannabinoids on β-arrestin2 recruitment. (C) Effects of phytocannabinoids on cAMP accumulation. (D) Effects of phytocannabinoids on β-arrestin2 recruitment. For cAMP accumulation experiments, HEK293 cells stably expressing GPR6 were treated with different concentrations of cannabinoids for 1 hour. Results are expressed as percent of basal cAMP accumulation. For β-arrestin2 recruitment experiments, CHO cells co-expressing both GPR6-PK1 and EA-β-arrestin2 were cultured for 48 hours and subject to stimulation with cannabinoids. Results are expressed as percent of basal relative luminescence units. Data shown represent the mean ± SEM of three experiments performed in quadruplicate. Significant differences from vehicle ( p < 0.05, one-way ANOVA with Bonferroni's post-test) are shown for *CBD, # Δ 9 -THC, † CBC, and & CBG. For phytocannabinoids, F-test results for each concentration were 0.1 μM: F (5,82) = 9.468, p < 0.05; 1 μM: F (5,90) = 18.69, p < 0.05; 10 μM: F (5,76) = 15.78, p < 0.05.

Article Snippet: The PathHunter TM Chinese hamster ovary (CHO)-K1 β-arrestin2 human GPR6 eXpress kits were purchased from DiscoverX (Fremont, CA).

Techniques: Stable Transfection, Expressing, Cell Culture, Concentration Assay

Journal: Heliyon

Article Title: Novel inverse agonists for the orphan G protein-coupled receptor 6

doi: 10.1016/j.heliyon.2018.e00933

Figure Lengend Snippet: CBD structure-activity relationship at GPR6. (A) Effects of CBD derivatives on cAMP accumulation. HEK293 cells stably expressing GPR6 were treated with different concentrations of CBD and CBD derivatives for 1 hour. Results are expressed as percent of basal cAMP accumulation. (B) Effects of CBD and CBD derivatives on β-arrestin2 recruitment. CHO cells co-expressing both GPR6 and EA-β-arrestin2 were cultured for 48 hours and subject to stimulation with CBD and CBD derivatives. Results are expressed as percent of basal relative luminescence units. Data shown represent the mean ± SEM of three experiments performed in quadruplicate. Significant differences from vehicle (p < 0.05, one-way ANOVA with Bonferroni's post-test) are shown for *CBD, § CBDV, † O-1821. F-test results for each concentration were 0.1 μM: F (4,65) = 17.06, p < 0.05; 1 μM: F (4,67) = 46.26, p < 0.05; 10 μM: F (4,64) = 59.93, p < 0.05.

Article Snippet: The PathHunter TM Chinese hamster ovary (CHO)-K1 β-arrestin2 human GPR6 eXpress kits were purchased from DiscoverX (Fremont, CA).

Techniques: Activity Assay, Stable Transfection, Expressing, Cell Culture, Concentration Assay

Journal: Heliyon

Article Title: Novel inverse agonists for the orphan G protein-coupled receptor 6

doi: 10.1016/j.heliyon.2018.e00933

Figure Lengend Snippet: Effects of synthetic cannabinoid agonists and antagonists on GPR6-mediated cAMP accumulation and β-arrestin2 recruitment. (A) Effects of synthetic cannabinoid agonists on cAMP accumualtion. (B) Effects of synthetic cannabinoid agonists on β-arrestin2 recruitment. (C) Effects of synthetic cannabinoid antagonists on cAMP accumualtion. (D) Effects of synthetic cannabinoid antagonists on β-arrestin2 recruitment. For cAMP accumulation experiments, HEK293 cells stably expressing GPR6 were treated with different concentrations of synthetic cannabinoids for 1 hour. Results are expressed as percent of basal cAMP accumulation. For β-arrestin2 recruitment experiments, CHO cells co-expressing both GPR6-PK1 and EA-β-arrestin2 were cultured for 48 hours and subject to stimulation with synthetic cannabinoids. Results are expressed as percent of basal relative luminescence units. Data shown represent the mean ± SEM of three experiments performed in quadruplicate. Significant differences from vehicle ( p < 0.05, one-way ANOVA with Bonferroni's post-test) are shown for *WIN55,212-2, # SR141716A and & SR144528. For synthetic agonists, F-test results for each concentration were 1 μM: F (3,54) = 5.813, p < 0.05; 10 μM: F (3,53) = 47.23, p < 0.05. For synthetic antagonists, F-test results for each concentration were 0.1 μM: F (2,58) = 4.721, p < 0.05; 1 μM: F (2,61) = 150.3, p < 0.05; 10 μM: F (2,61) = 359.9, p < 0.05.

Article Snippet: The PathHunter TM Chinese hamster ovary (CHO)-K1 β-arrestin2 human GPR6 eXpress kits were purchased from DiscoverX (Fremont, CA).

Techniques: Stable Transfection, Expressing, Cell Culture, Concentration Assay