FIGURE 5

Correlation of SNCG expression with inhibited DNMT3B expression by CSE in A549 cells. (a) Analysis of DNMT1 expression by western blot. A549 cells were treated with 5% CSE for 1–3 days. Total cell lysate was isolated at the indicated times and analysed for DNMT1 protein expression by Western blotting. (b) Analyses of DNMT3A and DNMT3B mRNA expression by real-time RT–PCR. A549 cells were treated with 5% CSE for a period of 0–72 h. At the indicated time points, total RNA was isolated and quantitative real-time RT–PCR with predeveloped probes to human DNMT3A, DNMT3B and GADPH was conducted to assess the mRNA levels. After normalization with GAPDH, the abundance of DNMT3A or DNMT3B mRNA in untreated cells was defined as 1, and the amounts of each mRNA from CSE-treated cells were plotted relative to that value. The figure shown is representative of three independent experiments in which each sample was assayed in triplicates. The results are mean s.d. (c) CSE withdrawal. A549 cells were untreated or treated with 5% CSE for 3 days. Then the fresh medium without CSE was added to cells, and the cells were harvested for genomic DNA and total RNA isolations at the indicated time points. The methylation status of SNCG was determined by MSP, the SNCG mRNA expression was examined by RT–PCR and the mRNA levels of DNMT3B were assessed by quantitative real-time PCR. DNMT3B mRNA in untreated cells was defined as 1, and the amounts of DNMT3B mRNA from CSE-treated cells at different time points after CSE withdrawal were plotted relative to that value. (d) Blocking DNMT3B expression with siRNA induces SNCG mRNA expression. A549 cells were transfected with DNMT3B siRNA or a scrambled RNA (as control) for 3 days. The mRNA levels of SNCG and DNMT3B were assessed by RT–PCR (inset) and real-time quantitative RT–PCR.