Figure 6.

ROCK activity is required to mediate the effects of stiffness on RPC proliferation, migration and differentiation. (a,b) Confocal images of F-actin immunodetection by phalloidin (red), paxillin (green) and nuclei with DAPI counterstain (white) in RPCs cultured on (a) 12 kPa and (b) 50 kPa substrate treated with vehicle (DMSO, control) and with the Rho kinase inhibitor Y27632. Bars = 25 μm (c) Average number of paxillin patches in RPCs cultured on 12 and 50 kPa substrates in control condition and treated with Y27632. At least 20 cells for each condition were analyzed. Box-and-whisker plots: Line = median, box = 25–75%, whiskers = 10–90%. (d) Percentage of growth inhibition induced by 10 μM Y27632 treatment of RPCs assessed by MTT assay. Data are mean ± SE (replicate n = 8). (e) MSD of RPCs cultured on 50 kPa stiffness in presence or absence of Y27632 (n = 46 and 44 trajectories for 50 kPa and 50 kPa + Y27632, respectively). (f) Percentage of inhibition of nephrin expression induced by 10 μM Y27632 treatment. (g) Percentage of inhibition of Klf15 expression induced by 10 μM Y27632 treatment. Data are expressed as mean ± SE. *p < 0.05 using one-way ANOVA followed by Tukey’s or Bonferroni post-hoc test, as appropriate.