Figure 4. p53 is required for GSK-3β regulation on let-7 activity.

(A) Forty-eight hours after transfecting BG-1 cells with Ctrl or GSK-3β siRNA as explained in Figure 2B, lysates were prepared to assess the levels of p53 and loading control GAPDH by western blot analysis. (B) Forty-eight hours after transfecting BG-1 cells with 50 nM Ctrl siRNA, GSK-3β siRNA, and/or p53 siRNA, lysates were prepared to measure protein levels (top) and cells in the culture media were collected and counted (graph). Signals were quantified by densitometry and normalized to the levels of β-actin (Fold). (C) Twenty-four hours after transfection as described in panel (B), BG-1 cells were transfected with pmirGLO-let7 and luciferase activity was measured 24 h later. (D) Twenty-four hours after transfection with Ctrl or GSK-3β siRNAs, HCT116 p53+/+ and p53−/− cells were transfected with pmirGLO-let7 and luciferase activity was measured 24 h later. Data in (B, C and D) are the means and S.D. from three independent experiments: * P<0.05; NS, not significant.