MKL1 localization is dependent on RhoA activation. (A) HEL cells were serum starved overnight and treated with TPA for the indicated times. Cell lysates were assayed for active Cdc42 (top), Rac1 (middle), and RhoA (bottom), as indicated. (B) Densitometric analysis of RhoA activation as a ratio of active RhoA to total RhoA confirmed activation of RhoA in HEL cells by TPA (representative of 3 independent experiments). (C) Time-lapse FRET images of 2 cells expressing a RhoA biosensor revealed an increase in RhoA activity after TPA treatment over the indicated time intervals. The relative RhoA activity scale is shown at right. (D) Serum-starved HEL-iMKL1 cells treated with TPA or calpeptin (a Rho activator) in the absence of TPA for the indicated time periods show that Rho stimulation is sufficient to drive nuclear accumulation of MKL1 (left). Serum-starved HEL-iMKL1 cells were pretreated with DMSO or cell-permeable C3 transferase (a Rho inhibitor) for 4 hours and then treated with TPA for 3 hours (right). Inhibition of RhoA decreased TPA-induced nuclear localization of MKL1. No fewer than 75 cells per time point were analyzed from 5 random fields of view in 3 independent experiments. Error bars indicate the SEM. n.s. indicates not significant. **P < .01.