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: Chemical Senses

Article Title: The stability of tastant detection by mouse lingual chemosensory tissue requires Regulator of G protein Signaling-21 (RGS21)

doi: 10.1093/chemse/bjab048

Figure Lengend Snippet: RGS21 binds gustducin-α and reduces bitterant signaling in a bitterant-responsive cultured cell line. (A) COS7 cell monolayers were transiently transfected with plasmid DNA encoding gustducin-α tagged with an HA epitope (“HA-Gα gust”). Forty-eight hours post transfection, cell monolayers were lysed in buffer containing GDP, Mg2+ and AlF4– (“AMF”) or GDP alone, as indicated. Clarified cell lysates were incubated with 10 μg of purified His6-RGS21 protein at 4°C overnight with NTA-agarose. Samples were centrifuged, agarose beads washed four times in lysis buffer, and precipitated proteins (“P”:) eluted by boiling for 5 minutes in loading buffer prior to being resolved by SDS–PAGE electrophoresis on the basis of molecular weight (“kDa” = kiloDalton), transferred to a nitrocellulose membrane, and detected using anti-HA antibody immunoblotting (“IB”:) and chemiluminescence. (B) Overexpression of wild-type RGS21, but not a loss-of-function, point mutant RGS21, leads to inhibition of bitterant signaling-induced reduction of cAMP levels. Cultures of the 16HBE cell line were transiently transfected with GloSensor cAMP biosensor cDNA and expression plasmids containing open reading frames for wild-type (“wt”) RGS21, or Arg-126-to-Glu point mutated (“R>E”) RGS21, or empty pcDNA3.1 vector, as indicated. Inhibition of forskolin-stimulated cAMP production (“100% maximum”; dotted line) by treatment with indicated concentration of the bitterant denatonium benzoate (i.e. EC50 previously established as per Supplementary Fig. S1) was determined 24 hr post-transfection by detection of GloSensor-dependent luminescence. Asterisks denote statistically significant differences as determined by one-way ANOVA with Bonferroni’s post-test: **, P < 0.01; ns, not significant (P >> 0.05).

Article Snippet: Briefly, monolayer cultures of the 16HBE cell line were transiently cotransfected using FuGENE6 (Promega) with the GloSensor cAMP-biosensor cDNA (Promega) and pcDNA3.1-based expression plasmids encoding either wild-type RGS21 open-reading frame, or a GAP-dead, loss-of-function point-mutant version (R126E a.k.a.

Techniques: Cell Culture, Transfection, Plasmid Preparation, Incubation, Purification, Lysis, SDS Page, Electrophoresis, Molecular Weight, Western Blot, Over Expression, Mutagenesis, Inhibition, Expressing, Concentration Assay

Journal: Cell

Article Title: Human Gain-of-Function MC4R Variants Show Signaling Bias and Protect against Obesity

doi: 10.1016/j.cell.2019.03.044

Figure Lengend Snippet:

Article Snippet: GloSensor cAMP biosensor - pGloSensor 20F plasmid , Promega , Cat#E1171.

Techniques: Virus, Recombinant, Plasmid Preparation, Software

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: Here, we used the live-cell biosensor GloSensor (Promega, Madison, WI, USA) to assess whether tumor β-ARs are coupled to the Gs-cAMP signaling pathway, that is, whether their stimulation causes an increase in cAMP levels.

Techniques: Stable Transfection, Expressing, Concentration Assay

Journal: Glia

Article Title: Glio‐ and neuro‐protection by prosaposin is mediated by orphan G‐protein coupled receptors GPR37L1 and GPR37

doi: 10.1002/glia.23480

Figure Lengend Snippet: TX14(A) acting on GPR37L1/GPR37 reduces cAMP levels in astrocytes. (a) Layout of the adenoviral vectors for knock‐down of GPR37L1 and GPR37. Each vector allows co‐cistronic expression of three pre‐miRNAs targeting different regions of the target gene. AVV = human adenoviral vectors serotype 5; CMV = human cytomegalovirus promoter; EmGFP = Emerald green fluorescent protein; miR155 = flanking pre‐miRNA sequence derived from miR‐155. (b) Western blot confirms that AVV–CMV–EmGFP–miR155/GPR37L1 and AVV–CMV–EmGFP–miR155/GPR37 (MOI 10) efficiently knock‐down GPR37L1 and GPR37 in astrocytes. AVV–CMV–EmGFP–miR155/negative is a control vector with hairpin sequence relevant to no known vertebrate gene. (c) Concentration–response curves for inhibition of cAMP production by TX14(A) in astrocytes pretreated with 1 μM NKH477. Cells were transduced with AVV–CMV–Glosensor and either AVV–CMV–EmGFP (control), a mixture of AVV–CMV–EmGFP–miR155/GPR37L1 and AVV–CMV–EmGFP–miR155/GPR37 to knock‐down GPR37L1/GPR37, or with AVV–CMV–EmGFP–miR155/negative ( n = 4, triplicates). (d) AVV–CMV–Glosensor transduced astrocytes were pretreated with PTX (20 hr, 100 ng/mL). About 100 nM TX14(A)‐induced cAMP reduction in astrocytes was PTX sensitive ( n = 12, *** p < .001 vs. indicated group, one‐way ANOVA with Turkey's post hoc analysis). (e): Astrocytes expressing an EPAC‐based cAMP sensor were kept in PSAP‐depleted media overnight and were stimulated with NKH477 (0.5 μM) in the absence or presence of TX14(A) (100 nM). TX14(A) decreased the transient cAMP signal; average of 58 astrocytes from four experiments. (f) Pooled data from (e) shows significantly decreased FRET ratio peaks with TX14(A) ( n = 58 = **** p < .0001, paired t test) [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: To assess intracellular cAMP changes, AVV were also used to express the Glosensor biosensor (Promega).

Techniques: Knockdown, Plasmid Preparation, Expressing, Sequencing, Derivative Assay, Western Blot, Control, Concentration Assay, Inhibition, Transduction