Figure 3. Bisulfite sequencing of the methylated pOct4-GFP plasmid recovered from HEK293 cells transfected in the absence or presence of the GADD45A expression vector.

A. The plasmid was methylated with HpaII methylase only. B. The plasmid was methylated with HpaII and HhaI methylases. The recovered DNA was treated with sodium bisulfite and the pOct4-EGFP target sequences containing the six HpaII sites (see Figure 2A) were amplified from bisulfite-treated DNA. The PCR products were cloned and between 22 and 25 individual molecules were sequenced. Closed circles mark methylated HpaII sites and open circles mark unmethylated HpaII sites. Overexpression of GADD45A (right panels) did not induce demethylation of the plasmid.