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. 2018 Aug 20;9:3320. doi: 10.1038/s41467-018-05837-7

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© The Author(s) 2018

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 — Local RGD density thresholds for adhesion formation and cell tethering to substratum. a dSTORM images of RGD ligands on ITO surfaces and paxillin in NIH-3T3 cells. NIH-3T3-cells-expressing paxillin fused to mEos2 were plated onto ITO surfaces that were functionalised with RGD-A647 and RGE peptides at the indicated ratios. Top: TIRF images of paxillin-mEos2; scale bar = 10 µm. Middle and bottom: Merged PALM images of paxillin-mEos2 (green) and dSTORM images of RGD-A647 peptides (red); scale bar = 10 µm. Zoomed regions of individual adhesions are shown in the second and third row; scale bar = 1 µm. Images are representative images of n = 3−4 independent experiments. b RGD density averaged over the entire surface (black bars) and averaged over the area occupied by cells (grey bars) obtained from dSTORM images of RGD-A647 on ITO surfaces with various ratios of RGD-A647:RGE. Bars and error bars are mean and standard deviation, respectively of n = 5 independent experiments; ns not significant; *P ≤ 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (paired t test). c Density of paxillin-mEos2 in adhesive structures (black bars) and outside adhesive structures (grey bars) in NIH-3T3 cells on ITO surfaces with various densities of RGD-A647:RGE. Paxillin-mEos2 densities in versus out of adhesive structures were significantly different on all surfaces (****P < 0.0001, unpaired t test) while paxillin-mEos2 inside adhesive structures was dependent on the RGD-A647:RGE ratio, except where indicated as ns, not significant (one-way ANOVA with Tukey post-testing). d Density of RGD-A647 in adhesive paxillin-mEos2 structures (black bars) and outside adhesive structures (grey bars) in NIH-3T3 cells on ITO surfaces with various densities of RGD-A647:RGE. RGD-A647 densities in versus out of adhesive structures were not statistically significant for all surfaces (unpaired t test, not shown) while RGD-A647 densities on 1:0 RGD:RGE surfaces was significantly higher in and out of adhesive structures compared to other presented (***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey post-testing). In b−d, data are average and standard deviation from n = 3−4 independent experiments