Figure 8.

Nuclear Rac1 activity is sufficient to rescue defects in Ect2-mediated transformed growth. (A) Rac1 is targeted to the nucleus upon disruption of its CAAX box and addition of an NLS. The localization of Ect2, NLS-Rac1-G12V-SAAX, and NLS-Rac1-F28L-SAAX was detected by confocal immunofluorescence microscopy, probing for HA-tagged Rac1 or Ect2 (green, Covance) and the nuclear marker DAPI (blue), n = 3. Scale bars represent 10 µm. (B) Stable expression of nuclearly targeted Rac1 mutants in Ect2 knockdown OVCAR8 cells. Lysate was immunoblotted with anti-HA (Covance) for ectopic Rac1 and Ect2 or with anti-Ect2 for endogenous + ectopic Ect2. Actin served as a loading control. (C) Rac1 mutant expression rescued the defect in anchorage-independent growth caused by Ect2 knockdown. Both constitutively active and fast-cycling nuclearly localized Rac1 mutants were able to rescue the defect in soft agar colony formation caused by Ect2 knockdown, to the same extent as re-expression of WT Ect2 (n = 3). Representative images (4× magnification) of colonies are shown. Scale bars represent 100 µm. Error bars represent SEM. Statistically significant differences are marked with * and ^ as in Figure 4C.