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: Frontiers in Pharmacology

Article Title: Inhibition of the Proliferation of Human Lung Fibroblasts by Prostacyclin Receptor Agonists is Linked to a Sustained cAMP Signal in the Nucleus

doi: 10.3389/fphar.2021.669227

Figure Lengend Snippet: MRE-269 and treprostinil, but not iloprost, cause sustained increases in nuclear cAMP. cAMP was measured in the nucleus of human lung fibroblasts transduced with the nucEpac2 FRET biosensor. Cells were stimulated with vehicle control (0.1% v/v DMSO) or a maximal concentration of MRE-269, treprostinil or iloprost (all 1 μM) (n = 5). (A) Time course of cAMP in the nucleus. (B) AUC calculated from A. (C) Representative ratiometric pseudocolour images from A. Cells were stimulated with vehicle control (0.1% v/v DMSO) or an EC 50 concentration of MRE-269 (200 nM), treprostinil (64 nM) or iloprost (25 nM) (n = 3). (D) Time course of cAMP in the nucleus. ( E ) AUC calculated from D. The arrow indicates addition of vehicle or IPR agonist at time 0. Maximal FRET change induced by the positive control (forskolin and IBMX) is indicated as a dashed line. (F) Time course of cAMP over 24 h in response to a maximal concentration of MRE-269, treprostinil or iloprost (n = 3). For time course graphs, symbols show the mean and error bars, standard error of the mean. For AUC graphs, bars show the mean, symbols the individual data points, and error bars show the standard error of the mean. For AUC graphs, * p < 0.05 and *** p < 0.001 vs. vehicle control, one-way ANOVA with Dunnett’s multiple comparisons test. For GloSensor time course graphs, ** p < 0.01 and *** p < 0.001 for MRE-269 vs. iloprost, ^ p < 0.05 and ^ ^ ^ p < 0.001 for treprostinil vs. iloprost, two-way ANOVA with Tukey's multiple comparisons test. For ratiometric images, scale bar shows 20 μm. MRE, MRE-269 and treprost., treprostinil.

Article Snippet: Human lung fibroblasts were electroporated with the GloSensor cAMP plasmid (Promega) using the 4D-Nucleofector System and P2 primary cell 4D-Nucelofector X Kit S (Lonza).

Techniques: Transduction, Control, Concentration Assay, Positive Control