DVL and GSK3 are required for Rac activation. (A) Quantitative PCR analysis of Dvl1 mRNA expression in leukemic MLL-ENL cells transduced with Scrambled (Sc) and Dvl1 (Dvl1a and Dvl1b) shRNA retroviral vectors. Bars represent means of quadruplicate values; and error bars, the SD. (B) Western blot analysis of Rac1 and Rac2 GTP and total Rac in leukemic MLL-ENL cells, transduced with Scrambled (Sc) and Dvl1 (Dvl1a and Dvl1b) shRNA retroviral vectors. At 48 hours after shRNA transduction, cells were selected with puromycin for 72 hours and then harvested for protein lysates. Data are representative of 3 independent experiments. (C) Number of colonies formed in methylcellulose by leukemic MLL-ENL cells transduced with Scrambled (Sc) and Dvl1 (Dvl1a and Dvl1b) shRNA retroviral vectors. Bars represent means of duplicate values; and error bars, the SD. **P < .01 versus control. (D) Western blot analysis of Rac1 and Rac2 GTP and total Rac in leukemic MLL-ENL cells, 24 hours after treatment with 2.5μM BIO acetoxime, 5μM CT99021, and 5μM SB216763. Data are representative of 6 (BIO acetoxime) and 3 (CT99021 and SB216763) independent experiments. (B,D) Numbers represent densitometric quantitation of Rac GTP levels normalized to total Rac protein bands. α-tubulin served as a loading control. (E) Number of colonies formed in methylcellulose by leukemic MLL-ENL cells treated with 2.5μM BIO acetoxime, 5μM CT99021, and 5μM SB216763. Bars represent means of duplicate values; and error bars, the SD. *P < .05, versus control. **P < .01 versus control.