Figure 3

PTEN inhibits BMI1 function. (A) DU145 cells were stably transfected with pBabe or pBabe-BMI1 retrovirus, followed by transiently transfected with pLHCX (empty vector) and pLHCX-PTEN retrovirus for 48 hours. The expression of FLAG-tagged BMI1, HA-tagged PTEN, p16^INK4A, p14^ARF, and actin was examined by western blot using specific antibodies. The relative p14^ARF and p16^INK4A expression was normalized against the respective actin and then expressed as fold changes of p14^ARF and p16^INK4A in DU145 cells co-infected with pBabe and pLHCX. The experiment was repeated at least three times by three individuals with identical results and representatives are shown. This information was presented under the p14 and p16 panels. Symbols * and ** show statistical significance (p < 0.05 and p < 0.01, respectively), in comparison to pBabe/pLHCX infected cells, determined by Student's t-Test (2-tails). (B) DU145 cells were stably transfected with pBabe and pBabe-BMI1 retrovirus, followed by assaying for hTERT activity using TRAP assay following the manufacturer's procedure. (C) 293T cells were transiently transfected with PTEN, PTEN(G129E) (G129E), PTEN(C124S) (C124S), C-terminal PTEN fragment (residues 186-403) (C-term), and BMI1 as indicated together with a hTERT promoter driven luciferase construct plus a β-Gal construct for 48 hours. Luciferase and β-Gal enzymatic activities were determined. Luciferase activities were normalized against β-Gal activities. Each transfection was carried out in triplicate and the experiment was repeated three times. **: p < 0.01 (in comparison to pcDNA); ++: p < 0.01 (in comparison to BMI1).