Requirement of ROCK-II but not ROCK-I for thrombin-induced EMP release. (A) Inhibition of EMP release by Rho-kinase inhibitors. Serum-starved HMEC-1 were preincubated 1 hour with different concentrations of Rho-kinase inhibitor Y27632 (left panel) or HA1077 (right panel) and then kept untreated or stimulated for 18 hours with 1 IU/mL thrombin in presence of the same quantities of inhibitors. EMPs were quantified in the cell-conditioned medium, as described in “Materials and methods.” Bar graphs represented mean values ± SEM for 5 independent experiments. *P < 0.5, **P < .01, ***P < .001. NS indicates not significant; control, unstimulated cells. (B) ROCK-II mediated EMP release by thrombin. Forty-eight hours after transfection with ROCK-II or ROCK-I or scrambled siRNA(s), HMEC-1 cells were kept untreated or were stimulated with 1 IU/mL thrombin for 18 hours, and EMPs were assayed in conditioned cell culture medium as above (right panel). Bar graphs represented mean values ± SEM for 3 independent experiments. *P < .05, ***P < .001; NS indicates not significant. Changes in the expression of ROCK-II and ROCK-I proteins were monitored by immunoblot 48 hours after transfection of HMEC-1 cells with ROCK-II or ROCK-I siRNA(s) (left panel).