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: The FASEB Journal

Article Title: Regionally selective cardiovascular responses to adenosine A 2A and A 2B receptor activation

doi: 10.1096/fj.202101945R

Figure Lengend Snippet: Glosensor cyclic AMP responses to CGS 21680 and BAY 60‐6583 in HEK293G cells. (A) Concentration‐response curves for agonist‐stimulated cAMP Glosensor luminescence responses in HEK293G cells in response to CGS 21680 and BAY 60‐6583. (B) The effect of simultaneous addition of 100 µM BAY 60‐6583 on the concentration‐response curve to CGS 21680, following 2 h pretreatment with PSB 603 (0.1 µM). Also shown is the effect of simultaneous addition of 10 µM SCH 58261 on the responses to CGS 21680 in the presence of PSB 603 (0.1 µM). (C, D) The effect of 100 µM BAY 60‐6583 on the response to 10 µM formoterol in the (C) absence and (D) presence of 0.1 µM PSB 603. (E) The effect of simultaneous addition of 10 µM SCH 58261 on the response to 10 µM formoterol. Data represent mean ± SEM of the peak luminescence response of 5 separate experiments, each carried out in triplicate. Data are expressed as a percentage of the peak luminescence response obtained with 100 µM NECA measured in the same experiment. ** p < .05 (unpaired t ‐test)

Article Snippet: A clonal HEK 293 cell line stably expressing the cAMP GloSensor (20F) biosensor (HEK293G) , was obtained from Promega (Madison, WI, USA).

Techniques: Concentration Assay