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. 2023 Sep 28;25(10):1453–1464. doi: 10.1038/s41556-023-01238-1

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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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Extended Data Fig. 3 — a, Fluorescence images of ITGβ5-GFP and plasma membrane marker RFP-CaaX on vitronectin-coated (top) and gelatin-coated (bottom) flat surfaces. Ratiometric images of ITGβ5/membrane (normalized by its mean per cell) are shown in the Parula colour scale. Scale: full-size, 10 µm; insets, 5 µm. b, Ethylenediaminetetraacetic acid (EDTA) treatment induces a dramatic reduction of ITGβ5-GFP accumulation at nanopillars in a live U2OS cell. Scale: full-size, 10 µm; insets, 5 µm. c, Quantification of the normalized ITGβ5/CaaX ratio at nanopillars and their surrounding flat regions. n = 994/711/994/711 nanopillars and their surrounding flat regions, from three independent cells. Medians (lines) and quartiles (dotted lines) are shown. P values calculated using paired Kruskal-Wallis test with Dunn’s multiple-comparison. d, Time-lapsed images of ITGβ5-GFP and CellMask Orange at Pillar 1 and 2 in Fig. 2e. At Pillar 1, the curved adhesion gradually assembles. At Pillar 2, the curved adhesion gradually disassembles. Scale: 1 µm. e, Fluorescence and bright-field images showing that immunolabelled ITGβ1 does not accumulate at vitronectin-coated nanopillars, while immunolabelled talin-1 does. Scale: full-size, 10 µm; insets, 5 µm. f and g, Fluorescence and bright-field images of immunolabelled talin-1 together with immunolabelled ITGβ1 (f) or ITGβ5 (g) on gelatin-coated nanopillar substrates. Talin-1 colocalizes with ITGβ1 in focal adhesions but does not accumulate at nanopillars. ITGβ5 appears mostly diffusive on gelatin-coated substrates. These results indicate that nanopillar-induced talin accumulation depends on ITGBβ5 and its ligands. Scale: full-size, 10 µm; insets, 5 µm. h, Fluorescence (YPet) and normalized ratiometric FRET images (in Parula colour scale) of the talin tension sensor N-terminal and C-terminal controls. Scale: full-size, 10 µm; insets, 5 µm. Source numerical data are available in source data.

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