Fig. 3

TRPV4 expression and activation is strongly correlated with volume expansion. a Western blot analysis and quantification of TRPV4 ion-channel protein expression (TRPV4) in MSCs cultured in hydrogel with varying stress relaxation and in fast-relaxing hydrogel with altered osmotic pressure for 7 days (**p<0.01 and *p<0.05, respectively, compared with fast stress relaxation and 0 kPa osmotic pressure case by one-way ANOVA test). b Representative images of immunohistochemical stainings of TRPV4 (yellow), actin (green), and nucleus (blue) of MSCs with different average volume of single cells. Arrows indicate a region of dense TRPV4 staining at cell periphery. Scale bar, 10 µm. c, d Representative images and quantification of intracellular calcium in MSCs cultured in hydrogel with varying stress relaxation (c) and in fast-relaxing hydrogel with altered osmotic pressure (d) for 7 days (****p<0.0001, ***p<0.001, and *p<0.05, respectively, compared with fast stress relaxation and 0 kPa osmotic pressure case by one-way ANOVA test, ^####p<0.0001 by Spearman’s rank correlation). Scale bar, 5 µm. All data are shown as mean ± s.e.m.