SUPPLEMENTARY INFORMATION - Nature Research€¦ · 2Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBERBBN), 08028 Barcelona, Spain

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Long-lived force patterns and deformation waves at repulsive epithelial boundaries

Pilar Rodríguez-Franco1, Agustí Brugués1, Ariadna Marín-Llauradó1,2, Vito Conte1, Guiomar Solanas3, Eduard Batlle3,4,5, Jeffrey J Fredberg6, Pere Roca-Cusachs1,7, Raimon Sunyer1,2*,

Xavier Trepat1,2,4,7*

Affiliations:

1 Institute for Bioengineering of Catalonia, The Barcelona Institute of Science and Technology, Barcelona 08028, Spain 2Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBERBBN), 08028 Barcelona, Spain 3Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, 08028 Barcelona, Spain 4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain. 5Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), 08028 Barcelona, Spain 6Harvard T. H. Chan School of Public Health, Boston, Massachusetts 7University of Barcelona, 08028 Barcelona, Spain

SUPPLEMENTARY MATERIAL

This document contains: - Supplementary Figures 1 to 10 - Supplementary Video legends 1 to 10

Long-lived force patterns and deformation waves at repulsive epithelial boundaries

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NMAT4972

NATURE MATERIALS | www.nature.com/naturematerials 1

http://dx.doi.org/10.1038/nmat4972

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SUPPLEMENTARY FIGURES

Supplementary Fig. 1: Characterization of EphB2 and ephrinB1 monolayers

a, Western Blot quantification of protein expression levels of EphB2 and ephrinB1 in the MDCK cell lines used in this study. These experiments show that EphB2-EcadhGFP cells express EphB2 but not ephrinB1, and that ephrinB1-EcadhCherry cells express ephrinB1 but not EphB2. b-e Immunostaining of EphB2 (b,c) and ephrinB1 (d,e) after monolayer collision. EphB2 and ephrinB1 localized at cell-cell junctions. Levels of EphB2 and eprhinB1 at the boundary were less pronounced than in cells behind it (see insets 1-4). Scale bar, 100 µm.





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Supplementary Fig. 2: Full view of a representative experiment

a-b Composition of phase contrast images (4×) of the EphB2 and ephrinB1 monolayers before collision (a) and after (b) collision. Right panels show magnification of the regions marked with a black square. Images are representative of n=2 independent experiments.





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Supplementary Figure 3. Oriented cell division during unjamming and jamming

a-c, Immuno-stainings of cell nuclei (top) and merged phalloidin (green), E-cadherin (red) and nuclei (blue) (bottom) at three different time points of boundary formation (6.5h (a), 11.5h (b) and 14.5h (c)). Scale bar is 45 µm. d-e, Percentage of cells dividing in stainings for the EphB2 (d) and ephrinB1 (e) sides of the monolayer. Divisions were distinguished among parallel (0±10º) or normal (90±10º) to the boundary. During maturation of the boundary (t=11.5h), divisions parallel to the boundary are dominant. Data are mean ± SE of n=4-16 experiments.





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Supplementary Figure 4. Experimental repeats show similar dynamics during the formation of EphB2/ephrinB1 boundaries

Kymographs of traction T⊥ (a), monolayer stress component σ⊥ (b) and velocity V⊥ (c) of an independent repeat during of the formation of an EphB2/ephrinB1 boundary. c, Autocorrelation function CVV⏊ of velocity kymographs for control cells during the jamming phase. Propagation velocity was 134 m/h.





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Supplementary Figure 5. Thymidine inhibited cell proliferation

a, Cell density in the monolayer at the first contact between the two opposing cell populations (t0), and 420 min later (t1) for control cells (ctr) and cells treated with thymidine. Data are mean ± SD of n=3 independent experiments.





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Supplementary Figure 6. Calcium chelation abrogates wave propagation

a, Kymograph of cell velocity V⊥ during collision of EphB2 and ephrinB1 monolayers. EGTA was added at 889 minutes and washed out at 938 minutes (the EGTA interval is labelled with dashed lines). The arrow head shows a wave front that becomes arrested shortly after EGTA addition. Data are representative of n=3 independent experiments. See also Supplementary Video 5.





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Supplementary Figure 7. Waves propagate long distances but attenuate before reaching the opposing edge.

a-c, Tiling of adjacent 20× images covering a large fraction of the horizontal extent of the two monolayers at different time points. t=0 is set as the contact time. d, Kymographs of velocity V⊥. Data are representative of n=3 independent experiments.





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Supplementary Figure 8: Blebbistatin impairs formation of a supracellular actin cable.

a-b, Stainings of phalloidin (green), E-cadherin (red) and nuclei (blue) at 6.5 h (a) and 15 h (b) after PDMS stencil release in blebbistatin treated cells. Images are maximum projections of spinning disk z-stacks. Scale bar is 55 m. c, Phalloidin and E-cadherin close-ups corresponding to the regions marked in a and b. Scale bar is 20 µm. d-e, Stainings of phospho-MLC (green), E-cadherin (red) and nuclei (blue) 15 h after PDMS stencil release for a control boundary (d) and a boundary under blebbistatin treatment (e). Images are maximum projections of spinning disk z-stacks. Scale bar is 55 µm. f, Phospho-MLC and E-cadherin close-ups of stainings shown in panels d and e. Scale bar is 20 µm.





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Supplementary Figure 9: Sustained elongation of cells at the boundary is a feature of the repulsive barrier between EphB2 and ephrinB1

Collision assays between homotypic monolayer pairs (EphB2/EphB2 and ephrinB1/ephrinB1, a-b) compared to the EphB2/ephrinB1 collision, c. For each collision, we segmented each boundary cell during the first contact between two opposing cells (t0), during full contact between the two monolayers (t1), and 250 minutes after full contact (t2). We then computed the average area, orientation and circularity (4π area/perimeter2) of each cell type. These results show that boundary cells at homotypic





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contacts relaxed their orientation and shape faster than cells at heterotypic contacts. Data are mean ± SE of n=3 experiments. Statistical tests were carried out to compare EphB2 cells in EphB2/ephrinB1 and EphB2/EphB2 assays, and ephrinB1 cells in EphB2/ephrinB1 and ephrinB1/ephrinB1 assays. *** P<0.001, Student’s t-test; NS, not significant.





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Supplementary Figure 10: Uncropped western blot scans from Supplementary Figure 1.





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SUPPLEMENTARY VIDEOS

Supplementary Video 1. EphB2/ephrinB1 monolayers form a repulsive interface

Collision assay of EphB2/ephrinB1 (left) and EphB2/EphB2 cells (right). 10× magnification. Scale bar is 175 µm.

Supplementary Video 2. Traction force evolution during the collision assay

Traction component Tx superimposed on EphB2/ephrinB1 phase contrast images. 20× magnification. Scale bar is 95 µm. Color bar is the same as in Fig. 2e-h.

Supplementary Video 3. Intercellular stress evolution during the collision assay

Intercellular stress component σxx superimposed on EphB2/ephrinB1 phase contrast images. 20× magnification. Scale bar is 95 µm. Color bar is the same as in Fig. 2i-l.

Supplementary Video 4. Velocity evolution during the collision assay

Velocity component Vx superimposed on EphB2/ephrinB1 phase contrast images. 20× magnification. Scale bar is 90 µm. Color bar is the same in Fig. 3e-h.

Supplementary Video 5. Calcium chelation abrogates wave propagation

Velocity component Vx superimposed on EphB2/ephrinB1 phase contrast images. 20× magnification. EGTA was added at 889 minutes and washed out at 952 minutes (labelled EGTA in the movie). Scale bar is 100 µm. See also Supplementary Fig. 6.

Supplementary Video 6. Waves propagate long distances before attenuating

Waves propagate long distances before attenuating. Velocity component Vx superimposed on EphB2/ephrinB1 phase contrast images. Tiling image acquisition at 20× magnification. Scale bar is 500 µm.

Supplementary Video 7. Inhibition of proliferation attenuates wave propagation

Velocity component Vx superimposed on EphB2/ephrinB1 phase contrast images of thymidine treated cells. 20× magnification. Scale bar is 75 µm. Color bar is the same in Fig. 4e-h.

Supplementary Video 8. Inhibition of contractility attenuates wave propagation





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Velocity component Vx superimposed on EphB2/ephrinB1 phase contrast images of blebbistatin treated cells. 20× magnification. Scale bar is 75 µm. Color bar is the same in Fig. 4m-p.

Supplementary Video 9. Collision between homotypic EphB2/EphB2 monolayers

Velocity component Vx superimposed on EphB2/EphB2 phase contrast images. 20× magnification. Scale bar is 100 µm.

Supplementary Video 10. Collision between homotypic ephrinB1/ephrinB1 monolayers

Velocity component Vx superimposed on ephrinB1/ephrinB1 phase contrast images. 20× magnification. Scale bar is 100 µm.





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SUPPLEMENTARY INFORMATION - Nature Research€¦ · 2Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina (CIBERBBN), 08028 Barcelona, Spain