Figure 6

Effect of 5-Fu on CASP8. (A) MCF-7, MB231, and SKBR3 cells were treated with or without 5-Fu (10 μM) for 3 days. RNA was isolated from the treated and untreated cells and RT-real-time PCR was performed with CASP8 and 18 s primers. Each bar represented the mean relative level of CASP8 and standard deviation (SD) from three measurements and normalized to 18 s. (B) DNA was isolated from the 5-Fu treated and untreated cells and modified with bisulfite treatment. MSP was performed with methylated and unmethylated CASP8 primers. The bands detected with methylated primer indicated methylated CASP8 (M), and the bands from unmethylated primer indicated unmethylated CASP8 (UM). (C) Bisulfite modified DNA from the indicated 5-Fu treated, and untreated cells were amplified with CASP8 primer designed according to the bisulfite modified sequence of CpG sites in CASP8 promoter as described in Methods. The amplified PCR products were purified and sequenced for confirmation of the CpG sites methylation. The dark dots in the bottom panel represented methylated CpG sites in the promoter, and open dots indicated unmethylated CpG sites.