Figure 2

5-aza-CdR restores Cav1 expression without affecting the hypomethylation of Cav1 CpG island (CGI). Cells were treated with 5 μM 5-aza-CdR for 6 days. Total RNA and genomic DNA were harvested for reverse transcription–PCR and bisulfite conversion. (a) Cav1 mRNA level measured by reverse transcription–PCR. The Cav1 level in DMSO treated cells was normalized to 1 (mean±s.e., n=3). **P<0.01. (b) UCSC genome browser view of Cav1 and distribution of CpG sites. The two two-headed arrows indicated the regions that are examined by methylation-specific PCR in (c) and (d). (c) CpG methylation on Cav1 CGI of MCF7 cell. Left: methylation-specific PCR result. UD, unmethylated DNA control. MD, methylated DNA control. U, results with primers specific for unmethylated sequence. M, results with primers specific for methylated sequence. Right: results of bisulfite genomic DNA sequencing covering the first 15 CpG sites on Cav1 CGI. Open squares indicate that CpG sites are fully unmethylated; partially filled squares indicate various degrees of CpG methylation. (d) CpG methylation on Cav1 5′-CGI shore. Left: methylation-specific PCR results of MCF7. Right: quantification of band density on the gel (mean±s.e., n=3). *P<0.05, **P<0.01.