Fig. 6.
Treatment of cells with EGCG or 5-aza-dc for 6 days reactivates silenced tumor suppressor genes, p16INK4a and Cip1/p21. (A and B) RNA was isolated from the cells of different treatment groups and subjected to the quantification of mRNA expression levels of p16INK4a and Cip1/p21 by real-time PCR using the procedure detailed in Materials and Methods. Data were normalized to housekeeping gene (β-actin) and are presented as relative change in mRNA levels in terms of mean values ± standard deviation (n = 3). Significant difference versus non-EGCG-treated control; *P < 0.001, **P < 0.01. (C) The protein levels of Cip1/p21 and p16INK4a were determined in cell lysates using western blotting under identical conditions. The relative density (arbitrary) of each band after normalization for β-actin is determined and shown as a fold-change compared with non-EGCG-treated control, which was assigned an arbitrary unit 1 in each case. Data under immunoblots show fold-change. Representative blots are shown from three experiments.