Figure 4

Reduction of transformed phenotype by Mel-18 overexpression and knockdown of BMI1 expression. A) Overexpression of Mel-18 in SGC-7901 cells results in increased cellular senescence (p < 0.05; upper panel: left, pictures of SA-β-gal stained cells; right, senescent cells were counted and plotted), decreased number of colonies in soft agar (p < 0.01, mid panel: left, pictures of colonies in soft agar, right, the number of colony were counted and plotted); and no difference in apoptosis (lower panel). The bars indicate mean ± SD. Vector infected cells served as a control. B) Knockdown of BMI1 in SGC-7901 results in increased cellular senescence (p < 0.05; upper panel: left, pictures of SA-β-gal stained cells, right, senescent cells were counted and plotted); decreased number of colonies in soft agar (p < 0.05; mid panel: left, pictures of colonies in soft agar, right, the number of colony were counted and plotted); and no difference in apoptosis (lower panel). Ctrli (non-specific sequence) served as control for BMI1 knockdown (BMI1i). C) Overexpression of Mel-18 leads to downregulation of BMI1, and reduction in pAKT (phospho-AKT) and upregulation of p16 expression as determined by Western blot analysis ( left, Mel-18, BMI1, pAKT, tAKT (total AKT), p16, and β-actin were detected by Western blot; right, quantification of the levels of BMI1 and p16, and AKT activity by densitometric analysis as described in Figure 3 ). D) BMI1 knockdown by RNAi approach leads to no change of Mel-18, reduction in pAKT and upregualtion of p16 expression as determined by Western blot analysis (left, pictures of Western blot; right, quantification of the levels of Mel-18 and p16, and AKT activity by densitometric analysis).