Figure 3

Effect of 5-aza-dc treatment on mRNA, protein expression and promoter methylation of CASP8 and maspin. Cells were treated with 5 μM 5-aza-dc for 3 days. (A) RNA were isolated from the treated and untreated cells and performed for RT-PCR with CASP8 primers. β-actin was used as loading control. (B) Total protein was isolated after cells were treated or untreated with 5-aza-dc. Western blot analysis was performed with antibodies specific for full length caspase-8, and β-actin was used as loading control. (C) DNA were isolated and modified by bisulfite treatment. MSP was performed with methylated and unmethylated CASP8 PCR primers as described in Methods. The bands detected by methylated primer represented methylated maspin (M), and the bands detected by unmethylated primer represented unmethylated maspin (UM). (D) The DNA from 5-aza-dc treated and untreated cells were modified by bisulfite treatment and 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 the CpG sites methylation. The dark dots represented methylated CpG sites in the promoter, and open dots indicated unmethylated CpG sites. (E) RNA from 5-aza-dc treated and untreated cells were used to perform RT and real-time PCR with maspin primers. The relative level of maspin gene was normalized to 18 s as described in Methods. Each bar represented the mean of three independent amplifications with standard deviation (SD). (F) DNA from cells treated and untreated with 5-aza-dc were modified by bisulfite treatment, and MSP was performed with methylated and unmethylated maspin PCR primers as described in Methods. The bands detected by methylated primer represented methylated maspin (M), and the bands detected by unmethylated primer represented unmethylated maspin (UM).