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Figure 4 | Molecular Cancer

Figure 4

From: Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced β-catenin/Tcf-Lef dependent transcription

Figure 4

Survivin effects on β-catenin protein levels and β-catenin/Tcf-Lef transcriptional activity were suppressed by inhibiting PI3K or Akt in HEK293T cells. A,B: HEK293T cells (5×105) were seeded in 6-well plates and transfected with pEGFP-C1 or pEGFP-survivin (1.5 μg). After transfection, cells were treated with LY294002 (20 μM) or wortmannin (10 nM). After 24 h, β-catenin, survivin, Akt, p-Akt, GFP and actin protein levels were evaluated by western blotting. Protein levels were quantified by scanning densitometric analysis of western blots and normalized to actin. C: HEK293T cells were seeded (5×105) in 6-wells plates and co-transfected with pcDNA or pcDNA-survivin (0.8 μg) and a dominant negative form (1 μg) of Akt (AktM). After 24 h, β-catenin, survivin, Akt, p-Akt and GAPDH protein levels were evaluated by western blotting. Protein levels were quantified by scanning densitometric analysis of western blots and normalized to GAPDH. D: HEK293T cells were seeded (5×105) in 6-wells plates and co-transfected with pEGFP-C1 or pEGFP-survivin (1.5 μg) and the reporter plasmids pTOP-FLASH or pFOP-FLASH (1 μg). After transfection, cells were treated with LY294002 (20 μM). Luciferase activity was obtained by calculating the pTOP-FLASH/pFOP-FLASH activity ratio for each condition. Data were previously normalized to values for β-galactosidase activity. E: HEK293T cells were seeded (5×105) in 6-well plates and transiently cotransfected with pcDNA or pcDNA-survivin (0.8 μg), the reporter plasmids pTOP-FLASH or pFOP-FLASH (1 μg) and in some cases dominant negative Akt (AktM, 1 μg). Luciferase activity was obtained by calculating the pTOP-FLASH/pFOP-FLASH activity ratio for each condition. Data were previously normalized to values for β-galactosidase activity. Numerical data shown are the means ± s.e.m. of results obtained in three independent experiments. Statistically significant differences compared to mock controls are indicated (* p < 0.05).

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