Journal: eLife

Article Title: Coalescing beneficial host and deleterious antiparasitic actions as an antischistosomal strategy

doi: 10.7554/eLife.35755

Figure Lengend Snippet: ( A ) Confocal images showing cell surface expression in mammalian HEK293 cells of 5-HT receptors cloned from S. mansoni ( Sm .5HTR L ), S. haematobium ( Sh. 5HTR) and S. japonicum ( Sj .5HTR) localized by COOH-terminally tagged eGFP. Scalebar, 50 µm. ( B ) Schematic of luminescent cAMP sensor bioassay. cAMP generated by schistosome 5-HTRs (blue) binds the engineered GloSensor luciferase, switching the sensor to a more active conformation resulting in enhanced luminescence output. ( C ) Kinetics of signal following the addition of 5-HT (1 µM) to HEK293 cells co-transfected with luminescent cAMP sensor and individual schistosome 5-HT receptors. Open circles, cells not transfected with 5-HT receptor, colored circles represent measurements in cells transfected with Sm .5HTR L (black), ( Sh. 5HTR (blue) and Sj .5HTR (red). ( D ) Serotonin dose-response curves for each of the three receptors (colored circles) and HEK293 cells expressing the cAMP sensor alone (open circles). Data reflect mean ± standard error of at least 3 biological replicates.

Article Snippet: Commercial assay or kit , GloSensor 22F cAMP sensor , Company , Promega; E2301 , .

Techniques: Expressing, Clone Assay, Bioassay, Generated, Luciferase, Transfection

Journal: eLife

Article Title: Coalescing beneficial host and deleterious antiparasitic actions as an antischistosomal strategy

doi: 10.7554/eLife.35755

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , GloSensor 22F cAMP sensor , Company , Promega; E2301 , .

Techniques: Recombinant, Software

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a Schematic of DREADD-β2AR and corresponding protein sequence encoding for signal peptide (black), VSV-G epitope (magenta), hM3Dq ligand binding domains (blue), and β2AR signaling domains (green). Black arrow: start of the mature GPCR after post-translational cleavage of the signal peptide. b Orthogonal view of DREADD-β2AR-transfected HEK cells immunostained for the N-terminal VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Schematic of signaling pathways for functional validation of the DREADD-based chimeras. The heterotrimeric G protein consists of an α- and βγ-subunit. Below: hM3Dq is a Gα q -coupled receptor, whereas non-chimeric β2AR recruits G proteins with a Gα s subunit. DREADD-β2AR contains the β2AR signaling domains to recruit Gα s . The DREADDs rM3Ds couples to Gα s and hM4Di is associated with Gα i . d Schematic of Gα s -coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. e – g Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-β2AR ( e ); non-chimeric β2AR ( f ), hM3Dq, rM3Ds or hM4Di ( g ); or empty vector ( e – f ). Baseline measurements of 30 min (first 15 min not shown) followed by ligand application (gray arrow for onset) of either CNO or levalbuterol. Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-β2AR: CNO 0.1–10 µM), seven (Empty vector: CNO), four (Non-chimeric β2AR: Levalbuterol; Empty vector: Levalbuterol; hM3Dq: CNO; hM4Di: CNO), or three (rM3Ds: CNO) experimental repetitions. Source data are provided as a Source Data file. h Schematic of Gα q -coupled GPCR engaging in the mitogen-activated protein kinase (MAPK) pathway which induces transcription of a firefly luciferase reporter from a serum responsive element (SRE). i Endpoint measurement of SRE-dependent luciferase activity in HEK cells transfected with hM3Dq (blue), DREADD-β2AR (magenta), non-chimeric β2AR (green), rM3Ds (red), hM4Di (cyan), or empty vector (gray). Ligand stimulation either with 10 µM CNO (left) or 0.001 µM levalbuterol (right). Dashed line: level of vehicle control. Error bars: standard error of the mean. Two-sided one-sample T-test for comparing to a mean of 1 representing the vehicle control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.004 (hM3Dq: CNO); p = 0.03 (rM3Ds: CNO); p = 0.001 (hM4Di: CNO); p < 0.002 (DREADD-β2AR: CNO); p = 0.09 (Empty vector: CNO); p < 0.001 (Non-chimeric β2AR: Levalbuterol); p = 0.01 (Empty vector: Levalbuterol). N = three experimental repetitions. Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Sequencing, Ligand Binding Assay, Transfection, Staining, Protein-Protein interactions, Functional Assay, Biomarker Discovery, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a Schematic of GPCR increasing baseline cAMP levels through constitutive activity. b Real-time measurement of cAMP-dependent luciferase activity during a 30 min baseline in HEK cells transfected with hM3Dq (blue), rM3Ds (red), hM4Di (cyan), DREADD-β2AR (magenta), or non-chimeric β2AR (green). Measure of center: Mean fold change compared to empty vector (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four experimental repetitions. Source data are provided as a Source Data file. c Graph shows average fold changes compared to empty vector control during the 30 min measurement in ( b ). Dashed line: level of empty vector control. Error bars: standard error of the mean. N = four experimental repetitions. Two-sided one-sample T -test for comparing to a mean of 1 representing the empty vector control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.81 (hM3Dq); p = 0.009 (rM3Ds); p = 0.48 (hM4Di); p = 0.01 (DREADD-β2AR); p = 0.009 (Non-chimeric β2AR). Source data are provided as a Source Data file. d Schematic of GPCR with constitutive activity impacting baseline MAPK signaling measured through an SRE reporter. e Endpoint measurement of SRE-dependent luciferase activity in transfected HEK cells. Dashed line: level of empty vector control. Error bars: standard error of the mean. N = seven (hM3Dq, DREADD-β2AR), four (rM3Ds, hM4Di), or nine (Non-chimeric β2AR) experimental repetitions. of four to nine repetitions. Two-sided one-sample T -test for comparing to a mean of 1 representing the empty vector control: *** p < 0.001; ** p < 0.01; n.s. p > 0.05. Exact p-values of individual T -tests without multiple testing correction: p < 0.001 (hM3Dq); p < 0.001 (rM3Ds); p = 0.30 (hM4Di); p < 0.001 (DREADD-β2AR); p < 0.001 (Non-chimeric β2AR). Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

doi: 10.1038/s41467-022-32390-1

Figure Lengend Snippet: a , b Orthogonal view of HEK cells transfected with DREADD-GPR65 ( a ) or DREADD-GPR109A ( b ) immunostained for the VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Top: Schematic of Gα s -coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Baseline measurement of 30 min (first 15 min not shown) followed by CNO application (gray arrow for onset). Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-GPR65) or seven (Empty vector) experimental repetitions. Source data are provided as a Source Data file. d Endpoint measurement of serum responsive element (SRE)-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Ligand stimulation with CNO. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T -test for comparing to a mean of 1 representing the vehicle control: * p < 0.05; n.s . p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.009 (DREADD-GPR65); p = 0.09 (Empty vector). N = four (DREADD-GPR65) or three (Empty vector) experimental repetitions. Source data are provided as a Source Data file. e Top: Schematic of Gα i -coupled GPCR reducing cAMP levels after ligand stimulation through adenylyl cyclase (AC) inhibition. Forskolin induces cAMP synthesis through AC activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Baseline measurements followed by application of CNO or vehicle (gray arrow for onset) and forskolin (white arrow for onset). Measure of center: Mean fold change compared to vehicle (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = five experimental repetitions. Source data are provided as a Source Data file. f Schematic of competition assay between Gα i -coupled DREADD-GPR109A and Gα s -coupled A2B adenosine receptor (A2BAR). Simultaneous stimulation of Gα i through CNO and Gα s through NECA prevents cAMP-responsive element (CRE)-mediated luciferase reporter activity. g Endpoint measurement of CRE-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Simultaneous stimulation with CNO and 5 µM NECA. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T -test for comparing to a mean of 1 representing the vehicle control: * p < 0.05; n.s. p > 0.05. Exact p -values of individual T -tests without multiple testing correction: p = 0.03 (DREADD-GPR109A); p = 0.34 (Empty vector). N = three (DREADD-GPR109A) or four (Empty vector) experimental repetitions. Source data are provided as a Source Data file.

Article Snippet: To measure Gα i -induced decreases in cAMP, we utilized a cAMP-dependent firefly luciferase suitable for Gα i signaling (GloSensor-22F; Promega; E2301) .

Techniques: Transfection, Staining, Activation Assay, Luciferase, Activity Assay, Plasmid Preparation, Control, Inhibition, Competitive Binding Assay