Figure 1

CASP8 promoter methylation resulted in decreased mRNA and protein expression. (A) DNA from the indicated cell lines were modified by bisulfite treatment, and MSP were performed with methylated and unmethylated CASP8 PCR primers as described in Methods. The bands detected by methylated primer represented methylated CASP8 (M), and the bands detected by unmethylated primer represented unmethylated CASP8 (UM). (B) RNA were isolated from cells and performed for RT and real-time PCR with CASP8. The relative level of CASP8 gene was normalized to 18 s mRNA as described in Methods. Each bar represented the mean of three independent amplifications with standard deviation (SD). (C) Total protein was isolated from the indicated cells, and Western blot analysis was performed with antibodies specific for full length caspase-8. β-actin was used as loading control. (D). Bisulfite modified DNA from the indicated 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 to confirm the location of methylated CpG sites. The dark dots represent location of methylated CpG sites in the promoter, and open dots indicate unmethylated CpG sites.