Journal: Journal of the Endocrine Society

Article Title: Real-Time Signaling Assays Demonstrate Somatostatin Agonist Bias for Ion Channel Regulation in Somatotroph Tumor Cells

doi: 10.1210/js.2018-00115

Figure Lengend Snippet: Effect of anti-G βγ ‒binding peptide on SS analogue signaling. Cells were incubated with (A) membrane potential dye, (B) cAMP GloSensor luciferin, or (C–E) intracellular Ca 2+ dye in the presence or absence of the G protein βγ subunit peptide blocker MPS-phosducin-like protein C-terminus (MPS-Phos, 1 µM). Cells were pretreated with MPS-Phos for 15 minutes at 28°C for cAMP GloSensor experiments or for 15 minutes at 37°C for the other experiments. (A) Hyperpolarization responses after simulation with SS14 (100 nM) or SS14 (100 nM) + MPS-Phos (1 µM). Treatment groups were not significantly different ( P = 0.5; two-tailed t test). (B) Inhibition of NKH477 (10 µM) stimulated cAMP production by SS14 (100 nM) or SOM230 (1 µM) in the absence or presence of MPS-Phos (1 µM). Response of MPS-Phos‒treated groups was not significantly different from that of nontreated groups (two-tailed t test). (C) HEK293-Sstr2A cells were incubated with the FLUO-8 am dye for 45 minutes, washed, and then incubated with the anti- βγ peptide MPS-Phos (2 µM) for 15 minutes. Cells were then treated with saturating concentrations of SS14 (100 nM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). Data are mean ± SEM from two different experiments, three replicates per group per experiment. (D) Intracellular Ca 2+ levels after stimulation with SS14 (100 nM) or SS14 (100 nM) + MPS-Phos (1 µM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). (E) Intracellular Ca 2+ levels after stimulation with SOM230 (1 µM) or SOM230 (1 µM) + MPS-Phos (1 µM). The effect of MPS-Phos was significant ( P < 0.0001; two-tailed t test). Data are expressed as a ratio of fluorescence intensity (F 1 /F 0 ). (A, B, D, and E) Data show the mean ± SEM from three independent experiments, three replicates per group per experiment. Black arrows indicate the addition of agonists 30 seconds after the start of the experiment. -, no ligand; SOM, SOM230; SS, somatostatin 14.

Article Snippet: Stable GH12C1 cell lines expressing a cAMP biosensor were created by transfecting the 22F GloSensor cAMP biosensor (Promega) into GH12C1-HA3-rSstr2A clone #35 cells using FuGENE (Promega) and selecting with 200 μg/mL of hygromycin B. Clonal cell lines were isolated by limiting dilution and were screened for biosensor activity.

Techniques: Incubation, Membrane, Two Tailed Test, Inhibition, Fluorescence

Journal: Toxins

Article Title: Human Peptides α-Defensin-1 and -5 Inhibit Pertussis Toxin

doi: 10.3390/toxins13070480

Figure Lengend Snippet: Human α-defensin-1 and -5 inhibit PT-mediated effects on cAMP signaling. PT (100 ng/mL) was pre-incubated with 12 µM α-defensin-1, α-defensin-5 or ß-defensin-1, or with the respective amount of solvent (H 2 O) for 15 min at room temperature and then added to iGIST sensor cells for 5 h at 37°C. For further control, cells were treated only with the solvent of PT. Then, inducing medium containing the luciferase substrate for the luminescent biosensor for cAMP was added. After 15 minutes of baseline measurement, cells were spiked with forskolin to activate adenylate cyclase and octreotide acetate to activate Gαi-coupled SSTR2 GPCR. Luminescence was recorded for further 60 minutes. ( a ) cAMP kinetic curves from one representative experiment are shown as mean ± SD (n = 3 from one experiment), con = cells treated only with forskolin (FSK) plus octreotide (Oct). ( b ) Bar graphs show baseline-subtracted area under the curve (AUC) from at least three independent experiments. Values are given as percent of PT-only-treated samples, mean ± SEM (n = at least nine from at least three independent experiments). Values for control samples are identical in both graphs. Results are shown in two separate graphs for better clarity. Significance was tested using one-way ANOVA followed by Dunnett’s multiple comparison test and refers to samples treated with PT only (left graph) or untreated controls (right graph) (** p ≤ 0.01,**** p ≤ 0.0001, ns not significant). ( c ) For control, iGIST sensor cells were treated only with FSK or with PT plus FSK in the absence of Oct to measure the maximal cAMP response in this assay. Values for control samples and samples treated with PT plus FSK and Oct are identical to values in ( a ).

Article Snippet: HEK293 cells (HEK-Gs/SSTR2_HA), ectopically expressing Gαi-coupled somatostatin receptor 2 (SSTR2) GPCR as well as a luminescence cAMP probe (GloSensor-22F, Promega, Mannheim, Germany) [ ], were cultivated in DMEM/F12 (containing glutamine and sodium pyruvate) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and penicillin–streptomycin (1:100) (Thermo Fisher Scientific).

Techniques: Incubation, Solvent, Control, Luciferase, Comparison

Journal: Toxins

Article Title: Domperidone Inhibits Clostridium botulinum C2 Toxin and Bordetella pertussis Toxin

doi: 10.3390/toxins15070412

Figure Lengend Snippet: DOM reduces the PT-mediated effects on cAMP signaling. Cells were treated with DOM at indicated concentrations and PT (100 ng/mL) for 5 h at 37 °C or cells were left untreated (con). Inducing medium with luciferase substrate for the luminescent cAMP biosensor was added. Baseline was measured for 15 min, then cells were spiked with forskolin (FSK, activator of adenylate cyclase) and octreotide acetate (Oct, activator of SSTR2). Luminescence was recorded for 60 min. ( a ) cAMP kinetic curves of one representative experiment are shown. Values are given as mean ± SD, n ≥ 3 from one experiment. ( b ) Bar graph shows baseline-subtracted area under the curve (AUC) from at least three independent experiments. Values are given as percent of samples treated with PT only, mean ± SEM, n ≥ 6. ( c ) As the control, cells were treated with FSK or PT plus FSK without Oct to detect maximal cAMP response. Values for the control samples and samples treated with PT, FSK, and Oct are identical to values in ( b ). Significance was tested by one-way ANOVA analysis and Dunnett’s multiple comparisons test, and values refer to samples treated with PT only (left graph in ( b , c ), white bar) or con (right graph in ( b ), white bar), (**** p < 0.0001, *** p < 0.001, * p < 0.05, ns = not significant p > 0.05).

Article Snippet: The iGIST bioassay [ ] is based on HEK293 cells that ectopically express the Gαi-coupled somatostatin receptor 2 (SSTR2) GPCR and a luminescent cAMP probe (GloSensor-22F, Promega, Madison, WI, USA).

Techniques: Luciferase, Control