Figure #4 – Nuclear envelope wrinkling depends on cytoskeletal-to-nuclear connectivity and regulates nuclear shuttling of mechano-sensitive transcriptional co-activators.

(A) (left) Images of YAP/TAZ immunostaining for representative MSCs with wrinkled or taut nuclei (as visualized by Lamin-A/C immunostaining). (A)(right) Nuclear-to-cytoplasmic ratios of YAP/TAZ from Figure #3E, where MSCs were cultured on five stiffness conditions with moduli ranging from 2.5 – 20 kPa for 18 hours (n = 101 nuclei over 5 stiffness conditions) using categorical wrinkling data separated by a wrinkling index cutoff of 5% (W = wrinkled regime, T = taut regime). Box plot represents median and 25^th/75^th quartiles (n=36–45 nuclei/group YAP/TAZ, Box plot represents median and 25^th/75^th quartiles, ^**** = p<0.0001 by Mann-Whitney U-test). SB = 5 μm. (B) Overview of experimental approach to reduce nuclear envelope to cytoskeleton connectivity via Nesprin 1 Giant (N1G) knockdown. (C) Representative XY maximum projection images and XZ slices of lamin-A/C immunostaining of MSCs three days post knockdown, with or without treatment with LPA for 30m prior to fixation and staining (SB = 5 μm). Corresponding quantification of the (D) wrinkling indices and (E) nuclear spread areas of the cells from part C (n> 34 cells/group, Box plot represents median and 25^th/75^th quartiles, * indicates p<005 and ^** indicates p<0.01 by Kruskal-Wallis one-way ANOVA testing with Dunn’s post-hoc). (F) YAP/TAZ nuclear ratios of MSCs three days post knockdown with negative control or N1G-targeting miRNA that where subsequently cultured on 5 or 55kPa polyacrylamide hydrogels for 18 hours prior to fixing and staining (n> 45 cells/group, Box plot represents median and 25^th/75^th quartiles shown, ^** = p<0.01, ^**** = p<0.0001 by Kruskal-Wallis one-way ANOVA testing with Dunn’s post-hoc).