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Image Search Results
Journal: Science (New York, N.Y.)
Article Title: A tool kit of highly selective and sensitive genetically encoded neuropeptide sensors
doi: 10.1126/science.abq8173
Figure Lengend Snippet: (A) Primary cultured rat cortical neurons expressing SST1.0 (A1) or CRF1.0 (A2), showing sensor expression (left), pseudocolor responses (middle), and cell membrane localization (right). Scale bars, 100 μm (left) and 20 μm (right). (B) Example fluorescence traces (left) and summary data (right) of neurons expressing SST1.0 (B1) or CRF1.0 (B2); where indicated, peptides and antagonists were applied (n = 66 to 115 ROIs from three or four coverslips). (C) Normalized dose-response curves of neurons expressing SST1.0 (C1) or CRF1.0 (C2) in response to the indicated ligands; n = 3 cultures each with 20 to 40 ROIs. (D) Summary of the fluorescence change measured in neurons expressing SST1.0 (D1) or CRF1.0 (D2) in response to a 2-hour continuous application of 1 μM SST-14 or 300 nM CRF, respectively; n = 4 cultures each with 20 to 40 ROIs. (E) Summary of normalized ΔF/F0 in HEK293T cells expressing SST1.0 (left) or CRF1.0 (right) in response to the indicated compounds applied: SST-14, CRF, UCN2, UCN3, CCK-8s, NPY, NTS, VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), adrenocorticotropic hormone (ACTH), β-melanocyte-stimulating hormone (β-MSH), γ-MSH, growth hormone-releasing hormone (GHRH), oxytocin (OT), vasopressin (AVP), vosotocin, isotocin, glucagon, glucagon-like peptide (GLP-1), DAMGO, dynorphin A (DynA), enkephalin (Enk), goserelin, ghrelin, galanin, orexin A (OX-A), SP, neurokinin B (NKB), and bombesin (BB) were applied at 1 μM, while Glu, GABA, DA, norepinephrine (NE), serotonin (5-HT), histamine (HA), ATP, ADP, adenosine (Ado), teriparatide (TPTD), and taltirelin were applied at 10 μM (n = 4 wells containing 100 to 300 cells per well). (F and G) G protein and β-arrestin coupling were measured using the split-luciferase complementation assay (F1), a cyclic adenosine monophosphate (cAMP) reporter (F2), and the Tango assay (G1 and G2) in cells expressing either the wild-type peptide receptor (red), sensor (green), or no receptor (Ctrl; gray) in the presence of the indicated concentrations of the ligand; n = 3 wells each. AU, arbitrary units. (H) RNA-seq analysis shows that GRAB peptide sensors did not alter the cellular transcriptome. Comparisons of transcriptomes between cortex tissue expressing SST1.0 or EGFP-CAAX (H1) and between CRF1.0 or EGFP-CAAX (H2). Pearson's correlation coefficient analysis was used to evaluate the differential RNA expression.
Article Snippet: Where indicated, the following compounds were applied to the cells in Tyrode’s solution by bath application or a custom-made perfusion system: SST-28 (Anaspec), SST-14 (Anaspec), CCK-8s (Abcam), CCK-4 (Abcam), CRF (Anaspec), UCNI (MedChemExpress), UCNII (MedChemExpress), UCNIII (Abcam), NTS (Anaspec), NPY (Abcam), VIP (Anaspec), PACAP(1–38) (MedChemExpress), PACAP(1–27) (MedChemExpress), Orexin-B (GL Biochem), Substance P (Tocris), Ghrelin (Tocris),
Techniques: Cell Culture, Expressing, Membrane, Fluorescence, Luciferase, RNA Sequencing, RNA Expression