Figure 4. p53 and endogenous miR-34 are functional for Axin2 suppression and nuclear GSK 3 levels. (A) UTR reporter assay and immunoblot analysis in wt vs. p53-null HCT116 cells. UTR reporter activities of Axin2 5′ UTR or/and 3′ UTR constructs were compared with wt p53 and p53^−/− isogenic HCT116 cells (left). The UTR reporter activities were significantly increased by loss of p53 function (*p < 0.01 compared with control, t-test). Immunoblot analysis of Axin2 and nuclear GKS3 levels in wt vs. p53-null HCT116 cells. Tubulin and HDCA1 are used as the loading control of whole-cell lysates and nuclear fraction, respectively. (B) UTR reporter (left), mature miR-34a (middle) and immunoblot analyses (right) after adenoviral transduction with a control (ad-GFP) or wt p53 (ad-p53) expression vector in p53-null HCT116 cells (*p < 0.01 compared with control, t-test). The endogenous Axin2 and nuclear GSK-3 levels were determined after transduction of adenovirus. (C) Inhibition of endogenous miR-34a increased activity of Axin2 UTR reporter in wt HCT116 cells (left, *p < 0.05 compared with control, t-test). Immunoblot analysis of Axin2 and nuclear GSK-3 level after transduction of negative control oligo (−) or anti-miR-34a (+) in HCT116 cells.