Figure 3.
Re-expression of AXIN2 and/or WIF1 inhibits WNT/β-CATENIN target gene expression through inactivation of AKT. A, Pfeiffer cells were transfected with either control (Ctrl) vector (pCMV-entry) or pCMV-entry/AXIN2 and/or pCMV-entry/WIF1 for 72 h, and mRNA levels of AXIN2, WIF1, CYCLIN D1, c-MYC, and SURVIVIN were analyzed by real-time RT-PCR. This experiment was conducted using three biological replicates with three technical replicates each, and β-ACTIN was used as control. The data shown represent the mean ± S.D., and ** indicates p values < 10−3. B, RIPA extracts (20 μg) prepared from either control-untransfected or -transfected Pfeiffer cells were analyzed by immunoblotting using the indicated antibodies. β-ACTIN was detected to show equal loading. C, levels of both active phospho-AKT (Thr-450 or Ser-473) and inactive AKT as well as active GSK3β or inactive phospho-GSK3β (Ser-9) were determined by immunoblotting using RIPA extracts (20 μg) prepared from Pfeiffer cells infected with lentivirus that expresses either control shGFP or shPRMT5 and Pfeiffer cells treated with either control DMSO or CMP-5. Both uninfected and CMP-6–treated Pfeiffer cells were used as internal controls. RIPA cell extracts were prepared 72 h post-infection or 48 h post-treatment. D, total RNA was prepared from Pfeiffer cells treated for 48 h with either control DMSO or increasing concentrations of AKT inhibitor IV (0.2 and 2 μm), and the mRNA levels of CYCLIN D1, c-MYC, and SURVIVIN were evaluated by real-time RT-PCR using gene-specific primers and probe sets as described in A. E, Pfeiffer cells were treated as in D, and RIPA extracts (20 μg) were analyzed by Western blotting using the indicated antibodies. β-ACTIN was detected to show equal loading.