FIGURE 1

SNCG expression and methylation status in A549 and H292 lung cancer cell lines. (a) SNCG mRNA expression in two lung cancer cell lines was examined by RT–PCR analysis. Two micrograms of total RNA was used in the reaction of reverse transcription in a volume of 20 l. Two microliters of the RT product was used in PCR with specific primers to SNCG or GAPDH. The RT–PCR products were separated on a 2% agarose gel and stained with ethidium bromide. SNCG protein expression in these two cell lines was examined by Western blot by using 50 g of protein of total cell lysate. MSP was used to assess the methylation status of SNCG CpG island in A549 and H292 cells. M, methylated PCR product; U, unmethylated PCR product. (b) The entire map of CpG island from each cell line was obtained by genomic sequencing as described in the 'Materials and methods' section. Positions are indicated relative to the translation start codon, and each circle in the figure represents a single CpG site. For each cell line, six independent clones were sequenced and each row represents one clone. , unmethylated; , methylated. (c) A549 cells were treated with 5  M of 5-aza-C for 4 or 6 days. Genomic DNA, total RNA and cell lysates were separated at the end of treatment. The methylation status of SNCG CpG island was determined by MSP; SNCG mRNA and protein expressions were determined by RT–PCR and Western blot analysis.