Fig. 2. The human RAR2 promoter is hypermethylated in MCF-7 breast carcinoma cells. A, bisulfite sequencing of the RAR2 promoter. The region analyzed is sketched (top), showing CpG sites (tick marks), the approximate position of the primers used (horizontal arrows) and the three CpG sites in the RARE (bracket) region. The RAR2 sequence (gi:9392669) was numbered relative to the transcription start site. Bisulfite sequencing results (bottom) are shown for each clone analyzed (, methylated CpGs; , unmethylated CpGs). Top panel, results for untreated cells; bottom panel, results for cells pretreated with azaC and treated with RA and TSA (see below); cells not pretreated with azaC and treated with RA and TSA yielded results similar to those for untreated cells (data not shown). B, semiquantitative RT-PCR analysis of RAR2 expression in untreated MCF-7 cells (Lane 1), cells treated with 1 µM RA and 100 nM TSA for 24 h (Lane 2), and cells pretreated with 5 µM azaC for 72 h and then treated with 1 µM RA and 100 nM TSA for 24 h (Lane 3).