Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2022 Aug 15;13:4728. doi: 10.1038/s41467-022-32390-1

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

PMC Copyright notice

Fig. 7 — a, b Orthogonal view of HEK cells transfected with DREADD-GPR65 (a) or DREADD-GPR109A (b) immunostained for the VSV-G tag under non-permeabilizing conditions. Magenta: VSV-G tag. Blue: nuclear staining with Hoechst. CMV, human cytomegalovirus promoter. c Top: Schematic of Gα_s-coupled GPCR inducing cAMP synthesis after ligand stimulation through adenylyl cyclase (AC) activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Baseline measurement of 30 min (first 15 min not shown) followed by CNO application (gray arrow for onset). Measure of center: Mean fold change compared to baseline mean (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = four (DREADD-GPR65) or seven (Empty vector) experimental repetitions. Source data are provided as a Source Data file. d Endpoint measurement of serum responsive element (SRE)-dependent luciferase activity in HEK cells transfected with DREADD-GPR65 (magenta) or empty vector (gray). Ligand stimulation with CNO. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T-test for comparing to a mean of 1 representing the vehicle control: *p < 0.05; ^n.s.p > 0.05. Exact p-values of individual T-tests without multiple testing correction: p = 0.009 (DREADD-GPR65); p = 0.09 (Empty vector). N = four (DREADD-GPR65) or three (Empty vector) experimental repetitions. Source data are provided as a Source Data file. e Top: Schematic of Gα_i-coupled GPCR reducing cAMP levels after ligand stimulation through adenylyl cyclase (AC) inhibition. Forskolin induces cAMP synthesis through AC activation. Below: Real-time measurement of cAMP-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Baseline measurements followed by application of CNO or vehicle (gray arrow for onset) and forskolin (white arrow for onset). Measure of center: Mean fold change compared to vehicle (dashed line) in the same experimental repetition. Ribbons: 95% confidence intervals. N = five experimental repetitions. Source data are provided as a Source Data file. f Schematic of competition assay between Gα_i-coupled DREADD-GPR109A and Gα_s-coupled A2B adenosine receptor (A2BAR). Simultaneous stimulation of Gα_i through CNO and Gα_s through NECA prevents cAMP-responsive element (CRE)-mediated luciferase reporter activity. g Endpoint measurement of CRE-dependent luciferase activity in HEK cells transfected with DREADD-GPR109A (magenta) or empty vector (gray). Simultaneous stimulation with CNO and 5 µM NECA. Dashed line: level of the respective vehicle control. Error bars: standard error of the mean. Two-sided one-sample T-test for comparing to a mean of 1 representing the vehicle control: *p < 0.05; ^n.s.p > 0.05. Exact p-values of individual T-tests without multiple testing correction: p = 0.03 (DREADD-GPR109A); p = 0.34 (Empty vector). N = three (DREADD-GPR109A) or four (Empty vector) experimental repetitions. Source data are provided as a Source Data file.