Figure 1
Survivin expression increased endogenous survivin and β-catenin protein levels, as well as β-catenin/Tcf-Lef dependent transcriptional activity: A,B: HEK293T cells (5×105) were transfected with pEGFP-C1 or pEGFP-survivin (A), pcDNA or pcDNA-survivin (B) or treated with SB216763 (20 μM), a pharmacological inhibitor of GSK-3β. β-catenin, survivin and actin protein levels were evaluated by western blotting. Protein levels were quantified by scanning densitometric analysis of western blots and normalized to actin. In panel A second lane, a band reflecting GFP dimerization is also visible. C,D: HEK293T cells (5×105) were seeded in 6-well plates and transiently cotransfected with pEGFP-C1 (1.5 μg) or pEGFP-survivin (0.5-1.5 μg) and additionally with the reporter plasmids pLuc-1710 (intact survivin promoter, 1 μg) pLuc-3M (survivin promoter with 2 TBEs mutated, 1 μg) (C), pTOP-FLASH (three functional TBEs in tandem) or pFOP-FLASH (all TBEs mutated, 1 μg each) (D). Luciferase activity was obtained by calculating the pLuc 1710/pLuc 3M or the pTOP-FLASH/pFOP-FLASH activity ratios for each condition. Data were previously normalized to values for β-galactosidase activity. E-H: NIH3T3 (E,F) cells (5×105) or MKN45 (G,H) cells (5×105) were seeded in 6-well plates and transiently transfected with pEGFP-C1 or pEGFP-survivin (1 μg each). E-G: β-catenin, survivin, GFP and actin protein levels were evaluated by western bloting. F-H: cells were cotransfected with pTOP-FLASH or pFOP-FLASH (1 μg) vectors and luciferase activity was obtained by calculating the pTOP-FLASH/pFOP-FLASH activity ratios 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).