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. 2021 Oct 28;12:6229. doi: 10.1038/s41467-021-26454-x

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© The Author(s) 2021

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 Schematic of the experimental protocol for assessing the role of HAVDI on reversing YAP nuclear localization in hMSCs. YAP accumulation in the nucleus was measured in hMSCs cultured on TCP (dark purple) in growth medium (pink) for 1, 3, 7, or 10 d, denoted DT1, DT3, DT7, and DT10, respectively. Reversal of YAP nuclear accumulation was assessed in hMSCs that were cultured on TCP in growth medium for these same time intervals, but then transferred (light yellow, day 0) to either soft (20 kPa) Scram/RGD (orange) or soft (20 kPa) HAVDI/RGD (blue) hydrogels for 1-10 d in growth medium before collection and analysis (gray, day 1–10). These conditions were denoted (DT1 + So1, 3), (DT3 + So1, 3), (DT7 + So1, 3, 10), or (DT10 + So1, 3, 5, 7, 10). Control cases included 3 d of cell culture on soft Scram/RGD (orange) or HAVDI/RGD (blue) hydrogels (So3), denoted “soft control (S/R)” and “soft control (H/R)”, respectively. b Representative immunostaining images of hMSC nuclei (blue) and YAP (green) after specified treatments on stiff and soft substrates as shown in a. Scale bars, 20 µm. S/R is Scram/RGD, H/R is HAVDI/RGD. c–e Quantification of YAP nuclear/cytoplasmic (n/c) ratios in hMSCs after specified treatments on stiff and soft substrates as shown in a. S/R (orange) and H/R (blue) represent all conditions related to soft Scram/RGD and HAVDI/RGD hydrogels, respectively. Soft control (S/R) and soft control (H/R) are baselines of YAP n/c ratios after 3 d culture for the S/R and H/R groups, respectively. YAP n/c ratio increased with culture time on TCP. c After 1 d of culture on TCP (purple, DT1), YAP localized to the nucleus (from left to right n = 108, 79, 70, 71, 108, 120, 100, 68 cells examined over 5, 11, 23, 15, 5, 26, 13, 13 images respectively, p values were obtained using one-way ANOVA followed by Tukey’s post hoc test, mean ± s.e.m.). Following transfer of hMSCs to either soft Scram/RGD (orange) or soft HAVDI/RGD (blue) substrates and culture for 1 d or 3 days (DT1 + So1, 3), the YAP n/c ratios decreased separately to the baseline of soft control S/R or H/R levels, showing a fully reversible YAP nuclear accumulation. d Following 3 d of culture on TCP (DT3), the YAP n/c ratio remained above the baselines of soft control in hMSCs transferred to Scram/RGD substrates for 1 d or 3 d (S/R: DT3 + So1, 3), but returned to the baselines in hMSCs transferred to HAVDI/RGD substrates for 1 d or 3 d (H/R: DT3 + So1, 3) (from left to right n = 108, 85, 93, 77, 71, 108, 85, 68, 72, 68 cells examined over 5, 6, 17, 15, 15, 5, 6, 19, 6, 13 images respectively, p values were obtained using one-way ANOVA followed by Tukey’s post hoc test, mean ± s.e.m.). This showed that, following 3 d of culture on TCP, YAP nuclear localization was partially reversible response without HAVDI (S/R), but fully reversible with HAVDI (H/R). e Following 10 d of culture on TCP (DT10), the YAP n/c ratio remained elevated and unchanged in hMSCs transferred to Scram/RGD substrates for up to 10 d (S/R: DT10 + So1, 3, 5, 7, 10), but decreased towards baseline levels within 1 d and maintained the levels in hMSCs transferred to HAVDI/RGD substrates for up to 10 d (H/R: DT10 + So1, 3, 5, 7, 10) (from left to right n = 108, 85, 66, 76, 74, 66, 78, 84, 84, 71, 108, 85, 66, 76, 70, 75, 102, 70, 78, 68 cells examined over 5, 6, 9, 11, 16, 21, 21, 23, 26, 15, 5, 6, 9, 11, 28, 21, 37, 20, 8, 13 images respectively, p values were obtained using one-way ANOVA followed by Tukey’s post hoc test, mean ± s.e.m.). This showed that, following 10 d of culture on TCP, YAP nuclear accumulation was permanent (irreversible) over the experimental timescale without HAVDI (S/R), but partially reversible with HAVDI (H/R). Source data are provided as a Source Data file.