Journal: International Journal of Molecular Sciences

Article Title: Crosstalk between β2- and α2-Adrenergic Receptors in the Regulation of B16F10 Melanoma Cell Proliferation

doi: 10.3390/ijms23094634

Figure Lengend Snippet: Effects of β-AR and a-AR ligands on cAMP production in B16F10 cells: ( A ) Representative image of cAMP measurements in real time using a GloSensor cAMP biosensor (bas, baseline). ( B ) Integrated cAMP responses computed as % of integrated forskolin response from tracings obtained in B16F10 cells stably expressing GloSensor-22F probe. Measurements were obtained in the absence or the presence of the β-AR agonist isoproterenol (ISO 1 μM), the antagonists propranolol and ICI 118,551 (PRO 10 μM, ICI 118,551 10 μM), isoproterenol plus either propranolol or ICI 118,551 (ISO 1 μM + PRO 10 μM or ICI118,551 10 μM), and the α2 agonists clonidine (CLO 1 μM) and ST91 (ST-91 1 μM). Data of three independent experiments are reported. * p < 0.05. ( C ) Concentration–response curves for the ligand-induced enhancement of cAMP production. Epinephrine (EPI), isoproterenol (ISO), norepinephrine (NE). EC 50 for ISO, EPI and NE was 4.6 nM, 45 nM and 284 nM, respectively.

Article Snippet: The luciferase-based intracellular cAMP probe GloSensor 22 F was purchased from Promega (Madison, WI, USA) and subcloned into puromycin resistance retroviral expression vector pQCXIP (Clontech, Mountain View, CA, USA).

Techniques: Stable Transfection, Expressing, Concentration Assay

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: eLife

Article Title: Inflammation produces catecholamine resistance in obesity via activation of PDE3B by the protein kinases IKKε and TBK1

doi: 10.7554/eLife.01119

Figure Lengend Snippet: ( A ) cAMP levels from 3T3-L1 adipocytes treated with or without 100 ng/ml TNFα for the indicated amount of time (left panel: 0–12 hr, right panel: 0–24 hr) followed by treatment with or without 25 μM FSK. **p<0.01. Performed in duplicate. ( B ) Relative gene expression (top panel: Ucp1 , bottom panel: Ikbke ) in 3T3-L1 adipocytes treated with or without 100 ng/ml TNFα for 24 hr followed by treatment with or without 10 μM ISO or 50 μM FSK, or 10 μM CL-316,243 for 4 hrs. ** P<0.01 and **p<0.0001. Performed in tripilicate. ( C ) cAMP levels as measured by Glosensor from 3T3-L1 adipocytes treated with or without 5 μg/ml poly (I:C) for 24 hr followed by treatment with or without 50 μM FSK over the course of 75 min. Performed in triplicate. ( D ) Glycerol release from 3T3-L1 adipocytes treated with or without different concentrations of poly (I:C) as indicated for 24 hr followed by treatment with or without 10 μM ISO or 50 μM FSK. ** p<0.0001. Performed in quadruplicate. ( E ) Immunoblots of whole cell lysates from 3T3-L1 adipocytes treated with or without different concentrations of poly (I:C) as same as for 24 hr followed by treatment with or without 10 μM ISO or 50 μM FSK. Results were replicated in multiple experiments. DOI: http://dx.doi.org/10.7554/eLife.01119.006

Article Snippet: 80 μl of packed 3T3-L1 cells was electroporated with 3 μg of Glosensor 22-F using Amaxa Cell Line Nucleofector Kit L (Lonza) according to the manufacturer’s protocol.

Techniques: Gene Expression, Western Blot