Figure 5. KapB activation of RhoA-GTPase but not the downstream kinase ROCK is necessary for p-body disruption.

A-D) HUVECs were transduced with recombinant retroviruses to express eGFP, a constitutively active version of Rho (RhoCA-eGFP), a dominant negative version of Rho (RhoDN-eGFP), KapB, or empty vector. 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 as described for Fig. 4. Alternately, cells were treated with the specific inhibitor of the Rho kinase ROCK 1/2 (10 µM of Y-27632) for one hour at 37°C before immunostaining. To quantify p-body disruption, the number of cells expressing KapB or controls that retained normal p-bodies was counted as for Fig. 4. 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 KapB 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 Fig. 4 (n = 2 independent experiments).