Figure 3.

Soft substrates promote hESC neuroepithelial conversion through a multi-targeted mechanotransductive process involving mechanosensitive Smad phosphorylation and nucleocytoplasmic shuttling regulated by rigidity-dependent Hippo-YAP activities and the actomyosin cytoskeleton (CSK). (a) Western blotting of total and phosphorylated Smad 1/5/8 (p-Smad 1/5/8) in hESCs cultured in neural induction medium for 3 d on rigid (E = 1,200 kPa) and soft (E = 5 kPa) PMAs. (b&c) Representative immunofluorescence images (b) and bar plot (c) showing rigidity-dependent subcellular localization of YAP in hESCs at day 0 and day 3. Scale bar in b, 50 μm. (d) Western blotting for YAP, TAZ, Smad 1/5/8, and Smad 2/3 in nuclear and cytoplasmic protein fractions from hESCs cultured for 3 d on rigid and soft PMAs. (e) Western blotting for phosphorylated YAP S127 (p-YAP S127) and YAP in whole cell lysates of hESCs after 3 d of culture on rigid and soft PMAs. (f&g) Representative immunofluorescence images (f) and bar plot (g) showing the percentage of Pax6⁺ NEs derived from scramble control and siLats1 knockdown hESCs after 6 d of culture on rigid and soft PMAs. Scale bar in f, 50 μm. (h) Bar graph showing the percentage of hESCs with nuclear localization of YAP after 3 d of culture with different drug supplementations, as indicated. (i&j) Representative immunofluorescence images (i) and bar plot (j) showing the percentage of Pax6⁺ NEs derived from hESCs on rigid and soft PMAs after 6 d of culture with different drug supplementations, as indicated. Scale bar in i, 50 μm. In Western blots β-actin and GAPDH were used as protein loading controls and lamin A/C for nuclear fraction control. Data represents the mean ± s.e.m with n ≥ 3. P-values in c & g and h & j were calculated using two-side unpaired student t-tests and one way ANOVA followed by Tukey post hoc analysis, respectively. *, P < 0.05; **, P < 0.01.