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. 2020 Oct 28;28(4):1193–1207. doi: 10.1038/s41418-020-00643-5

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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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Fig. 3 — a Graphical representation of the experimental setup used to assess pluripotent stem cell (PSC) response to changes in physiological substrate stiffness. YAP–TEAD-mCherry hESC reporter cells were cultured onto soft surface (0.5 kPa) and then moved to surfaces with increasing stiffness (2, 20 and 64 kPa). b Representative FACS plots depicting mCherry fluorescence in YAP–TEAD-mCherry hESC reporter cells cultured for 48 h on substrates with physiological stiffness (n = 3). c Boxplot representation of the Elastic Modulus of CTR and YAP−/− hESCs grown onto substrates with increasing stiffness (2, 20 and 60 kPa). The values were obtained by AFM and are expressed as Pascal (Pa) (n = 12, *P < 0.05, one-way ANOVA test followed by Holm-Sidak’s test for multiple comparison). d Left: representative confocal images depicting F-actin (green) cytoskeleton arrangement in isogenic (CTR) and YAP−/− hESCs (YAP−/−) grown onto substrates with increasing physiological stiffness. Right: barplot representation of the intensity of green channel (F-actin) in isogenic (CTR) and YAP−/− hESCs (YAP−/−) grown onto substrates with increasing physiological stiffness (n = 3). e Left: orthogonal sections from Z-stack confocal images showing the basal (left) and apical (right) distribution of F-actin in CTR, YAP−/− and YAP −/− hESCs in which YAP has been re-expressed (RESCUE). F-actin is stained with Phalloidin (green) and nuclei counterstained with DAPI (blue). Side views show sagittal sections of the monolayered cells. Right: 3D Z-stack reconstruction and cross-sectional view of the perinuclear actin of CTR, YAP−/− and RESCUE hESCs (n = 3). The images were obtained by IMARIS software after staining with Phalloidin (F-actin, green) and DAPI (nuclei, blue). f Orthogonal sections from Z-stack confocal images showing F-actin organization (green) in CTR and XAV939-treated hESCs for 48 h. Side views show sagittal sections of the monolayered cells. F-actin was stained with Phalloidin (green) and nuclei were counterstained with DAPI. g Boxplot representation of the elastic modulus of CTR, YAP−/− and RESCUE hESCs (n = 12, ****P < 0.0001; *P < 0.05, Kruskal–Wallis test followed by post hoc Dunn’s test for multiple comparisons). h Schematic representation of the strategy followed to discover proteins involved in cytoskeleton organization which are regulated by substrate stiffness through YAP in pluripotent stem cells (PSCs). i Volcano plot representation of differentially regulated genes in CTR versus YAP−/− hESCs grown on substrates with physiological (0.5 and 64 kPa) and tissue culture polystyrene (TCPS). (n = 3, P < 0.05, log2 Fc < |0.58|). j Venn diagram representation of differentially regulated genes in CTR versus YAP−/− hESCs grown on substrates with physiological (0.5 and 64 kPa) and tissue culture polystyrene (TCPS). k Venn diagram representation of PSC YAP bona fide targets that have an annotation for cytoskeleton organization (GO: 0007010) and found dysregulated onto substrate with controlled stiffness (0.5 and 64 kPa) and Tissue culture polystyrene (TCPS). l Volcano plot representation of cytoskeleton-bound proteins significantly regulated in YAP−/− compared to CTR hESCs, as identified by TMT Mass Spectrometry. (n = 5, P < 0.05, log2 Fc > |0.58|). l Left: barplot representation of cytoskeleton-bound proteins significantly regulated in YAP−/− compared to CTR hESCs, as identified by TMT mass spectrometry, that were defined as YAP bona fide targets with cytoskeleton annotation (GO:0007010). Right: identification of TEAD1-binding sites in selected YAP targets with cytoskeleton organization annotation. m Dotplot representation of elastic modulus in CTR and YAP−/− hESCs treated or not with F-actin polymerizing agent jasplakinolide for 24 h as obtained by AFM analysis. (n = 12, ****P < 0.0001, Kruskal–Wallis test followed by post hoc Dunn’s test for multiple comparisons).