Akt kinase activity is required for the E2 -induced reinitiation of the cell cycle progression. A: MCF-7 cells seeded in 35 mm dishes were transfected with shRNA Akt (1 + 2) (0.5 μg per dish) together with expression vectors of shRNA-resistant mutants of Akt1 (Akt1R) or Akt2 (Akt2R) as indicated, and an indicator plasmid encoding luciferase cloned downstream of cyclin A promoter (0.5 μg). A β-galactosidase vector (100 ng) was included to allow the correction for transfection efficiency. For more details see Materials and Methods. The cells were lysed and the activity of luciferase and β-galactosidase were determined. The results shown are means and s.e.m. of triplicates. B: The cells were transfected as in A with shAkt (1 + 2), Akt2R, or kinase-dead shRNA-resistant mutants of Akt1 (Akt1R/KD) or Akt2 (Akt2R/KD) as indicated. After transfection, the cells were made quiescent as in A. To a set of dishes of each transfection mix, E2 was added to a final concentration of 1 μM, the remaining dishes were left in the medium containing ICI 182780 (control). The data presented show the induction factor (E2 vs. control) calculated for the luciferase/β-galactosidase activities. The results shown are means +/− s.e.m. of triplicates.