Upstream activators of Rho GTPase (MK2 EE and hsp27 DDD) disrupt p-bodies.

<p>A–D) HUVECs were transduced with recombinant retroviruses that express a constitutively active version of MK2 (MK2 EE, Flag-tagged) or phosphomimicking heat shock protein (hsp)27 (hsp27 DDD, HA-tagged). Following two-day selection with puromycin, cells were seeded onto coverslips. The next day, cells were either not treated or treated with C3 transferase (in basal media) for 6 hours at 37°C to irreversibly inactivate Rho GTPase. After the 6-hour incubation, cells received one hour of normal media before fixation, permeabilization, and staining with the following primary antibodies: mouse anti-hedls or rabbit anti-DDX6 (p-bodies, green), rabbit anti-HA (hsp27 DDD-HA, false-colored blue), or mouse anti-Flag (MK2 EE-Flag, false-colored blue). To visualize actin stress fibers, cells were also labeled with phalloidin (red). To quantify p-body disruption, the number of cells expressing MK2 EE or hsp27 DDD that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a>. n = 3 independent experiments Scale bar = 10 µm. E) HUVECs were sequentially transduced with two populations of recombinant retroviruses: firstly, puromycin-resistant viruses that express MK2 EE-Flag, hsp27 DDD-HA or the empty vector; and secondly, blasticidin-resistant viruses that express a dominant negative version of Rho (Rho DN-HA tagged) or the empty vector. At each step, positive transductants were selected with the appropriate drug for 2 days. Following this, cells were seeded onto coverslips and the next day, treated with basal media for 1 hour before fixation, permeabilization and staining as described. To quantify p-body disruption, the number of cells expressing the transgene of interest that retained normal p-bodies was counted as for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004597#ppat-1004597-g004" target="_blank">Fig. 4</a> (n = 2 independent experiments).