Fig 2: Rho/ROCK signaling regulates miR-145/KLF4 to mediate the cell elongation-induced MSC-to-SMC differentiation.(A) MSCs were cultured on flat or 10 µm microgrooved substrates and treated without or with C3 or Y-27632 for 24 h, followed by stainings with FITC-phalloidin for cell morphology and DAPI for nuclei. Scale = 20 µm. (B) Quantification of total cell area, cell body aspect ratio, nuclear area and nuclear aspect ratio in the four groups were shown. Data are presented as the mean ± SEM, with 35–55 cells in each group. * vs. flat control, ***P < 0.0001. # vs. 10 µm microgrooved control, #P < 0.05, ##P < 0.005, ###P < 0.0001. & vs. C3 treated group, &P < 0.05, &&P < 0.005, &&&P < 0.0001. (C) Western blot analyses of a-SMA, SM-22 and GAPDH in MSCs treated with C3, Y-27632 or the vehicle control and cultured on flat or microgrooved substrates. The graphic data are mean ± SEM of band intensity normalized to flat control, n = 3. *P < 0.05. (D) MiR-145 expression in MSCs treated with C3, Y-27632, or the vehicle control and cultured on flat or microgrooved substrates. n = 3. * vs. flat control, **P < 0.005. # vs 10 µm microgrooved control, ##P < 0.005. (E) qRT-PCR analysis of KLF4 gene expression in MSCs treated with C3, Y27632, or the vehicle control and cultured on flat or microgrooved substrates. n = 3. * vs. flat control, **P < 0.005. # vs 10 µm microgrooved control, #P < 0.05, ##P < 0.005. (F) Western blot analyses of a-SMA, SM-22, and GAPDH in MSCs treated with C3 or Y27632 and transfected with PM145 or PMC control and cultured on flat or microgrooved substrates. The graphic data are mean ± SEM of band intensity normalized to flat control, n = 3. * vs. PMC in C3 treated group, *P < 0.05; # vs. PMC in Y-27632 treated group, #P < 0.05.