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. 2020 Mar 24;10:5356. doi: 10.1038/s41598-020-62202-9

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© The Author(s) 2020

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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Viral particle distance to CD151 and integrin maxima. (A–C) Membrane sheets were generated from HaCaT cells treated for 5 h with EdU-PsVs. They were stained for EdU-PsVs (cyan lookup table; click-labelled with fluorescein), CD151 (green lookup table; immunostaining) and integrin α6 (red lookup table; immunostaining), followed by STED microscopy imaging. (A) Images from the three channels and an overlay are shown and displayed at arbitrary intensity scalings (linear lookup tables). Circles mark identical pixel locations. (B) Pearson correlation coefficient between PsVs and CD151 (left) and integrin α6 (right). A control value for randomized distribution (‘flipped’) was calculated after flipping of one channel. (C) For individual PsVs, we plotted their nearest distance to a CD151 and an integrin α6 maximum (the analysis includes 4452 PsVs from 57 membrane sheets collected from three biological replicates). (D–F) As in (A–C) but cells were analysed and we stained for integrin α3 (the plot in F includes 8519 analyzed PsVs from 60 cells collected from three biological replicates). The pattern of PsV binding to the cell membrane looks the same on cells and membrane sheets (Fig. S10). Values are given as means ± SD (n = 57 membrane sheets or 60 cells, each collected from three biological replicates). Statistical analysis was performed employing the unpaired Student’s t-test comparing the original to the flipped images (***p < 0.001). Red boxes frame viral particles potentially used for further analysis of the platform area (Fig. 5). Only PsVs were considered that were closer than 250 nm to bright CD151/integrin maxima.