Fig. 5. WNT3a stimulates activation of small-molecular-weight GTPases, while expression of dominant-negative (DN) versions of small-molecular-weight GTPases or treatment of rFZ1-expressing cells with specific siRNAs targeting the same small GTPases abolish WNT3a stimulation of JNK activity. (A) F9 cells expressing rFZ1 were treated with WNT3a (100 ng/ml) for 10 minutes and activation of each of the small-molecular-weight GTPases was monitored as described in the Materials and Methods. (B,C) F9 cells expressing rFZ1 were transfected (1 µg/well in a 12-well plate) with dominant-negative versions of small-molecular-weight GTPases (B) or treated with siRNAs specific for RhoA, Rac1 and Cdc42 (C) for 24 hours and JNK activity was determined. (D) F9 cells stably expressing rFZ1 were transfected with DVL3-GFP2 alone or together with DN-RhoA for 24 hours, and JNK activity was determined. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots. The DVL3-RhoA epistasis experiment is representative of two independent experiments whose results were in strong agreement. The results displayed for the activation of small-molecular-weight GTPases are from a single experiment performed with triplicate sampling. These results are a representative of two separate experiments whose data were in high agreement. **P<0.01 versus –WNT3a control; ^##, P<0.01 versus +WNT3a control.