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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. 9 — a, Anti-vitronectin staining illustrates the fibre morphology of IMR-90 lung fibroblast-derived ECM. Z-depth is colour-coded. Scale: 10 µm. b, In 3D ECM made of vitronectin fibres, colocalization of AF647-collagen with immunolabelled vitronectin confirmed the incorporation of vitronectin in ECMs. Scale: full-size, 50 µm; insets, 25 µm. c, 3D super-resolution reconstruction of AF647-labelled collagen fibres. Top left: the widefield diffraction-limited image. Right: the z projection of the 3D super-resolution reconstruction. Yellow arrows point to some individual collagen fibres. Colour encodes the z position. Scale: 5 µm. d, Extracting the diameter of individual fibres. (i) The image shows an example of a resolved individual fibre. Transverse line profiles are taken across the fibre to estimate its diameter. (ii) Localizations from the line profile are binned into a histogram which is then fit to a Gaussian function (orange line). The full width at half maximum is extracted to estimate the diameter. Scale: 1 µm. e, Top and side views (3D projection) of a thick 3D ECM made of AF647-labelled pure collagen fibres. Z depth is colour coded. Scale: 10 µm. f, Representative 3D images (x-z projection) of U2OS cells expressing GFP-CaaX, 72 hrs after being plated on the top of a matrix made of pure collagen fibres (left) or vitronectin fibres (right). Scale: 10 µm. g, x-y image (Z0 plane) and x-z image (Y0 plane) of z-stack images showing the U2OS cells expressing GFP-CaaX in vitronectin fibres, presented in f (right side). Scale: 10 µm. h, Zoom-in x-y images of the area indicated in g showing plasma membrane folding along vitronectin fibres at the middle (z = Z0), bottom (z = Z1), and top (z = Z2) of cells. This result suggests that cell plasma membranes are deformed by 3D ECM fibres. Scale: 10 µm.