Figure 4. Ad-lnc-p21-MRE suppresses the activity of Wnt/β-catenin pathway in ALDH+ CSC in a miR-451-regulated manner.

A. ALDH⁺ CSCs were infected with 10 MOI of Ad-EGFP, Ad-lnc-p21 or Ad-lnc-p21-MRE. Cell lysate was collected 48 hrs later and the levels of β-catenin in nuclear extracts and whole cell lysate (WCL) were determined by western blot. Lamin B and GAPDH served as loading controls for nuclear lysate and WCL, respectively. B. NCM460 cells were transduced as in (A) The cells were co-transfected with the mixture of TOPFlash reporter plasmids and pRL-TK control plasmids (1 μg / 1 × 10⁶ cells; 40:1), and/or Wnt 3a-expressing plasmid (500 ng / 1 × 10⁶ cells) and miRNA-451 inhibitor (50 nM) for 48 hrs. TOPFlash reporter activity was examined to quantify the activity of β-catenin signaling. pRL-TK was used as a transfection control. C. ALDH⁺ CSCs were infected with indicated adenoviruses (10 MOI). The cells were co-transfected with the mixture of TOPFlash reporter plasmids and pRL-TK control plasmids (1 μg / 1 × 10⁶ cells; 40:1), with or without miRNA-451 inhibitor (50 nM). Forty eight hours later, TOPFlash reporter activity was determined. D. mRNA levels of β-catenin-responsive target genes, Axin2 and Lgr5, were detected in the ALDH⁺ CSCs with infections. GAPDH was used as an endogenous control. Representative images (A) are shown. Data are presented as the mean ± SD (C, D, E) of each group from triple replicates. **P < 0.01.