Spry2 is DNA hypermethylated, and its expression is repressed in mouse B-cell lymphoma lines but can be reactivated by treatment with a demethylating agent. (A) GBS of a CpG-rich region located between −728 and −484 in the Spry2 promoter in mouse B-cell lines 3-1, Wehi, Bal 117, and A20. Each row represents the sequence of an individual clone. ○ represents unmethylated CpG sites; ●, methylated sites; and squares, 2 CpG loci within the Not1 site. (B) Spry2 expression was assayed by quantitative PCR in 3-1, Wehi, Bal 117, and A20 mouse B-cell lines. N.D. indicates not detected. Spry2 expression is normalized to 36b4 (Rplp0) expression. DNA methylation status for each tumor cell line was also assessed by MSP. The top and bottom bands correspond to PCR products amplified by primers specific for methylated (M) or unmethylated (U) CpG sites after bisulfite conversion. (C) Spry2 expression assayed in triplicate without and with 5-aza-dC and/or 100 nm TSA treatment.