Fig. 1: Development and in vitro characterization of NOPLight.
From: Development of a genetically encoded sensor for probing endogenous nociceptin opioid peptide release
a Structural model of NOPLight and screening summary. n ≥ 3 cells for each variant. b Representative images of HEK293T cells (top; scale bars, 10 µm) and neurons (bottom; scale bars, 20 µm) expressing NOPLight before and after application of 1 µM N/OFQ and ΔF heat map. c Responses of NOPLight or NOPLight-ctr in HEK293T cell and neurons to 1 µM N/OFQ, followed by competition with 10 µM J-113397. d Quantification of maximal responses from (c), n = 38, 30, 22, 27 cells from three independent experiments, respectively. Data shown as mean ± SD. (One-sided Mann–Whitney U-test, ****P < 0.0001, P = 1.003 × 10−12, and 1.262 × 10−9 for NOPLight versus NOPLight-ctr in HEK293T and neurons). e Normalized response of HEK293T cells and neurons expressing NOPLight to N/OFQ titrations (mean ± SD) fitted with three-parameter Hill equation. n = 3 independent experiments per concentration. f Line-scan time plot of NOPLight ΔF/F0 (gray) from membrane-pixels shown in bottom. Right, quantification of time constant (τ) from four independent experiments. Data represented as mean ± SD. g Left: experimental schematics and images (scale bars, 20 µm), right: time plot of normalized ΔF/F0 from a representative experiment (gray) with quantified time constant (τoff). Data represented as mean ± SD. h Maximal NOPLight response to: NCS, Nocistatin; J11, J-113397; UFP, UFP-101; RO, Ro 64-6198; MCO, MCOPPB; OFQ1-11, Orphanin-FQ (1–11)). *P < 0.05, **P < 0.01, P = 0.024, Nocistatin; 0.0012, J-113397; 0.0012, UFP-101. n = 9 cells except MCO (n = 8) from three independent experiments. i Maximal NOPLight response to endogenous opioid ligands (1 µM). **P < 0.01, P = 0.003, Dynorphin A; P = 0.003, Dynorphin-B; P = 0.003, Leu-Enkephalin; P = 0.003, Met-Enkephalin; P = 0.003, β-endorphin. n = 9 from three independent experiments. Statistical comparisons in (h, i) by one-sided pairwise Mann–Whitney rank test with post hoc Bonferroni correction, data represented as mean ± SEM. j NOPLight response to neurotransmitters (DA dopamine, ACh acetylcholine, GABA gamma-Aminobutyric, all 1 mM). n = 29 (DA) or 30 (ACh, GABA) cells from three independent experiments (two-sided paired t-test, ****P < 0.0001, P = 2.45 × 10−22, 7.46 × 10−19, and 7.82 × 10−21, respectively).
