Figure 5

t-DARPP-induced cell growth is dependent on PI3K/AKT signaling pathway. A, MCF-7 cells stably expressing t-DARPP or pcDNA3.1 vector were treated with either vehicle or 40 μM of LY294002 for 30 min or 2 h. The cells were washed with PBS and grown for 24 h, and then subjected to Cell-Titer-Glo Luminescent Cell Viability Assay. The results indicated a significant increase of cell growth in t-DARPP-expressing cells relative to control cells after treatment with vehicle. This growth increase was almost completely abrogated after treatment with LY294002. B, MCF-7/t-DARPP and MCF-7/pcDNA3.1 cells were treated with vehicle and 40 μM of LY294002 for 30 min or 2 h, and then grown for 24 h. Protein extracts were subjected to western blot analysis of t-DARPP, p-AKT^ser473, and AKT. The results showed that p-AKT^ser473 protein level was significantly higher in t-DARPP-expressing cells than control cells, and treatment with LY294002 blocked phosphorylation of AKT^ser473 in all cells. C, MCF-7/t-DARPP and MCF-7/pcDNA3.1 cells were transfected with scrambled siRNA or Akt siRNA and grown for 48 h. The cells were then subjected to Cell-Titer-Glo Luminescent Cell Viability Assay. The results indicated a significant increase of cell growth in t-DARPP-expressing cells relative to control cells after transfection with scrambled siRNA. This growth increase was partially abrogated after transfection with Akt siRNA. D, MCF-7/t-DARPP and MCF-7/pcDNA3.1 cells were transfected with scrambled siRNA or Akt siRNA and cultured for 48 h. Protein extracts were analyzed by Western blot for p-AKT^ser473, AKT, t-DARPP, and β-actin. Results are representative of four experiments and shown as the mean ± SD. Significance of difference was calculated using Student's t test analysis.