Extended Data Fig. 3. Signaling characterization of nLightG and nLightR.
a, Intracellular calcium signaling recorded in HEK293T cells co-expressing nLightG (left) or the swAlpha-1 AR (right) along with the red fluorescent calcium indicator jRGECO1a. Fluorescence response of the calcium indicator was measured at baseline conditions, upon addition of NE (10 μM final concentration) and upon addition of ionomycin (10 μM final concentration), as indicated by the colored bars. Signals were normalized to the maximum response of the same cells after addition of 10 μM ionomycin. n= 19 and 22 cells for swAlpha-1 AR-jRGECO1a and nLightG-jRGECO1a. b, Statistical analysis of the responses shown in (a) for nLightG. Individual data points represent the mean ΔF/F0 response of jRGECO1a for each cell upon addition of NE. Violin plot represents the kernel density estimate of the probability density function for each sample. Mean values before and after addition of NE were compared using a two-tailed Students t-test with Welch’s correction. P = 2.887x10-7. c, Same as in a but for nLightR in combination with the green fluorescent calcium indicator GCaMP6s. n = 21 and 23 cells for swAlpha-1 AR-GCaMP6s and nLightR-GCaMP6s, respectively. d, Same as in b but for the data shown in c for nLightR in combination with GCaMP6s. P = 3.393x10-21. e, Time traces of the luminescence ratio of HEK293T cells co-expressing either nLightG (green traces), the swAlpha-1 AR (grey traces) or the hmDRD1 (blue traces) fused to SmBiT and a mini G-protein or β-arrestin-2 fused to LgBiT. NE (10 μM final concentration) or FluoroBrite DMEM was added to the cells between 490 s and 520 s. The luminescence ratio between stimulated and non-stimulated cells was calculated and normalized to the baseline luminescence ratio before the addition of NE. Each trace is the average of three independent experiments. f, Same as in e but for nLightR (magenta traces). g, Statistical analysis of data shown in e and f. The mean luminescence ratios before and after the addition of NE were calculated and compared using a two-tailed Students t-test with Welch’s correction. P values were as follows: mini-Gq, swAlpha-1 AR, 7.75×10-3; nLightG, 0.590; nLightR, 0.589; mini-Gs, swAlpha-1 AR, 3.88×10-3; nLightG, 0.281; nLightR, 0.410; mini-Gi, swAlpha-1 AR, 0.126; nLightG, 0.792; nLightR, 0.147; β-arrestin-2, swAlpha-1 AR, 8.97×10-2; nLightG, 0.134; nLightR, 0.377. All data are shown as mean ± SEM and all experiments were repeated three times with similar results.