Treatment of human skin cancer cells, A431 and SCC13, with GSPs or 5-aza-dc for 5 days reactivates silenced tumor suppressor genes, p16INK4a, Cip1/p21 and RASSF1A. (A, B and C) RNA was isolated from the cells of different treatment groups and subjected to the quantification of mRNA expression levels of tumor suppressor genes using real-time PCR using the procedure detailed in Materials and methods. The mRNA levels were normalized to housekeeping gene (β-actin) and are presented as relative change compared to non-GSPs-treated controls, which was assigned an arbitrary unit 1 in each case. The mRNA levels are expressed in terms of mean values ± S.D. from three independent experiments. Significant difference versus non-GSPs-treated controls, *P<0.001, **P<0.01. (D) The levels of tumor suppressor proteins, RASSF1A, Cip1/p21 and p16INK4a, were determined in cell lysates using western blotting under identical experimental conditions. Representative blots are shown from three experiments.