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. 2023 Jun 8;19(9):1147–1157. doi: 10.1038/s41589-023-01350-1

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© The Author(s) 2023

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/.

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Fig. 4 — a, Schematic representation of ChemoG–NAD. b, Normalized fluorescence intensity emission spectra of SiR-labeled ChemoG–NAD at different NAD⁺ concentrations. Means of three technical replicates are shown; [NAD⁺], NAD⁺ concentration. c, NAD⁺ titration curves of ChemoG–NAD labeled with different fluorophores. Data are shown as the means ± s.d. of the FRET/eGFP ratio changes (ΔR/R₀; n = 3 technical replicates). ΔR/R₀ and C₅₀ values are summarized in Supplementary Table 8. d, NAD⁺ titration curves of ChemoX–NAD_SiR biosensors. Data are shown as the means ± s.d. of the FRET/eGFP ratio changes (ΔR/R₀; n = 3 technical replicates). The intracellular free NAD⁺ concentration range is indicated with a gray box. ΔR/R₀ and C₅₀ values are summarized in Supplementary Table 8. e, Confocal images of U-2 OS cells expressing ChemoG–NAD labeled with SiR. Shown are the eGFP channel, the FRET channel and the ratio image of both channels (FRET/eGFP) in pseudocolor (LUT = mpl-viridis). Cells were treated for 24 h with DMSO (Ctrl), 100 nM FK866 or 1 mM NR; scale bars, 25 µm. f, Dot plots representing the FRET/eGFP ratios of ChemoG–NAD_SiR expressed in U-2 OS cells treated as described in d; n = 133 (Ctrl), 117 (NR) and 132 (FK866) cells from three independent experiments. P values are given based on unpaired two-tailed t-test with Welch’s correction; ****P < 0.0001. Data are shown as the means ± s.d. g, Confocal image of U-2 OS cell coexpressing ChemoB–NAD-cyto and ChemoG–NAD-mito labeled with SiR. Shown are the FRET donor FP channels, the FRET channels and the composites of the FP or FRET channel of both sensors pseudocolored (eBFP2 (cyan), eGFP (green), eBFP2-FRET (orange) and eGFP-FRET (magenta)). The brightness of the donor and FRET channels was adjusted to show potential cross-talk between the channels; scale bars, 25 µm. h, Time course measurement of ChemoB–NAD-cyto (cytosol) and ChemoG–NAD-mito (mitochondria) fluorescence intensity coexpressed in U-2 OS cells and labeled with SiR. Represented are the means of the FRET/FP ratios (line) and single-cell traces (dim lines) normalized to 1 at t = 0 min. The addition of MNNG is indicated with an arrow (n = 28 cells from four biological replicates).

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