ROCK inhibitor Y-27632 reverses effects of MK2-K339R. (A) HUVECs were transduced with the appropriate adenoviral constructs (Ad-LacZ, Ad-WT-MK2, Ad-DN-MK2, or Ad-K339R) for 24 hours. Confluent monolayers were wounded as in Figure 6, pretreated with the ROCK inhibitor Y-27632 (10μM) for 1 hour, and wound closure with or without TNF-α (10 ng/mL) recorded as in Figure 6. Quantification for the percentage wound closure can be seen on the right. Note the lack of effect of Y-27632 on vehicle wound closure, but that the ROCK inhibitor reversed TNF-α–mediated inhibition of EC migration. Also note the reversal effect of Y-27632 on the Ad-MK2-K339R–mediated inhibition of EC migration under TNF-α stimulation. (B) HUVECs were transduced with Ad-LacZ or Ad-MK2-K339R, stimulated by TNF-α (10 ng/mL) for 0-60 minutes with or without Y-27632 pretreatment, and then harvested for immunoblotting. Note that Y-27632 had no effect on MK2 expression, MK2 activation, or HSP27 phosphorylation. (C) HUVECs were prepared under the same conditions as in (B), stimulated with or without TNF-α (10 ng/mL) for 6 hours, and stained with Alexa-Fluor 488 phalloidin. Note the loss of elongated ECs under TNF-α stimulation when Y-27632 was added. Moreover, Y-27632 caused a loss of hyperelongated ECs in the Ad-MK2-K339R–transduced culture. (D) Quantification of cell elongation performed as in Figure 4E. Panels A-D show representative images from 1 of 3 independent experiments and quantitative data (n = 3). Values are means ± SEM. *P < .05; **P < .01.