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. 2016 May 18;6:26113. doi: 10.1038/srep26113

Figure 5. The molecular pathway by which ROCK inhibitor improves cell adhesion onto a substrate.

Figure 5

(a) Phase contrast images show MCECs seeded onto culture plates after 3 hours. (b–d) MCECs seeded without or with Y-27632 were stained with phalloidin and an antibody for vinculin. The mean amount of vinculin per cell was evaluated. The mean cell area was smaller in control MCECs than in Y-27632-treated MCECs. *P < 0.01, **P < 0.05. (e) MLC was phosphorylated in control cells, even after 24 hours, while MLC phosphorylation was suppressed in MCECs treated with Y-27632. (f) Cell dissociation was induced by EGTA treatment, and phosphorylation of MLC was evaluated by immunostaining. (g) MCECs were seeded on non-adhesion culture plates or adhesion culture plate, and phosphorylation of MLC was evaluated by western blotting. MLC phosphorylation was sustained in MCECs seeded on non-adhesion culture plates, while it was suppressed in MCECs seeded on adhesion culture plates. (h–j) MCECs were seeded and expression of vinculin and phosphorylated MLC was evaluated by immunostaining. Control MCECs expressed phosphorylated MLC without expression of vinculin, whereas phosphorylation of MLC was suppressed in MCECs treated with a ROCK inhibitor (Y-27632) and an MLC inhibitor (blebbistatin) associated with expression of vinculin. *P < 0.01, **P < 0.05. (k) MCECs were seeded and the effect of inhibition of MLC on focal adhesion molecule activity was evaluated by western blotting. (l) MCECs were seeded with or without blebbistatin and adhered numbers of MCECs were evaluated with the CellTiter-GloTM luminescent cell viability assay after 24 hours. (m) Cell dissociation was induced by EGTA treatment, and activity of RhoA was evaluated by a pull-down assay. GTP-bound active RhoA was highly expressed in dissociated MCECs. (n) MCECs were seeded with or without C3 (a Rho inhibitor) and the numbers of adhered MCECs were evaluated by the CellTiter-GloTM luminescent cell viability assay after 24 hours. (o) The involvement of integrins on cell adhesion enhancement by Y-27632 was evaluated by seeding MCECs in the presence or absence of integrin-neutralizing antibodies, and the numbers of adherent cells were determined. *P < 0.01, **P < 0.05.