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. 2023 Apr 1;6:358. doi: 10.1038/s42003-023-04706-4

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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. 5 — a–d FRET/FLIM images and analysis for COS-7 cells expressing a PIEZO1-mTurquoise2 plus CDH5-mVenus (CDH5-mVenus) or b PIEZO1-mTurquoise2 plus VGFR2-SYFP2. a, b Intensity images (white, high intensity), lifetime images calibrated to the rainbow scale indicated at the top corner (3.5–4.2 ns) and graphs of the lifetime distributions in which grey vertical lines indicate the rainbow scale limits. Scale bars, 50 µm, apply to all images. c, d Box plot summary peak whole cell lifetime data for the experiment types of a and b. 3 independent experimental repeats for PIEZO1-mTurquoise2 alone (-CDH5/VGFR2, black) and PIEZO1-mTurquoise2 plus CDH5-mVenus (***P = 7.68 ×10⁻⁴) or VGFR2-SYFP2 (P = 0.72393) (+CDH5/VGFR2, red). Superimposed data points are average lifetime values for individual images (−CDH5, N = 9; +CDH5, N = 9; −VEFGR2, N = 9; +VGFR2, N = 9). e–j Images and image analysis for retinal veins of HA-PIEZO1 mice (PIEZO1^HA) or wild-type (WT) mice (PIEZO1^WT) immuno-stained with αCDH5 antibody (green) and αHA antibody (grey). e, f Representative images for αCDH5 and αHA staining with merger of these images to the right. Scale bars, 20 µm. g, h are the line-intensity (grey value) plots for the vertical scan lines superimposed on the Merge images of e and f. Green αCDH5, grey αHA. Light grey highlighting indicates cell-cell junctions. i, j Box-plot quantification of image intensity for e PIEZO1^HA or f PIEZO1^WT retinal veins stained with αHA, shown for junctional regions indicated by αCDH5 staining (+CDH5) and non-junctional regions (−CDH5) in arbitrary fluorescence units (AFU). The intensity of each image was normalised to the image background. **P = 0.00347. Data are for n = 3 independent experiments. The superimposed data points are the average intensity for individual images, PIEZO1^HA (−CDH5, N = 13 and +CDH5, N = 13) PIEZO1^WT (−CDH5, N = 10 and +CDH5, N = 10).