Effect of decitabine on global DNA methylation (5mC and 5hmC) and Dnmt genes in differentiating neural stem cells (NSC). (A) Schematic representation of decitabine treatment. Briefly, 2.5 μM of decitabine was added to dissociated neurospheres on day 0 (D0) at the onset of NSC differentiation for 48 h, and the treatment was withdrawn at D2. Cells were kept in culture till D8. Top panel (B-E), after exposure to decitabine at D2. (B) Immunofluorescent detection of DNA methylation using 5-methylcytosine (5mC) antibody. Decitabine caused reduced levels of DNA methylation, note the presence of 4',6-diamidino-2-phenylindole (DAPI) signals in decitabine-treated cells with no 5mC signal. Scale bars represent 5 μm. (C) Detection of overall DNA methylation levels by DNA dot blot with antibodies specific for (a) 5mC, (b) 5-hydroxymethylcytosine (5hmC). (D) Quantification of the 5mC and 5hmC levels after decitabine exposure. (E) Detection of Dnmt transcript levels by qRT-PCR. Bottom panel (F-I), after withdrawal of decitabine at D8. (F) DNA methylation detection by immunofluorescence using 5mC antibody. Scale bars represent 5 μm. (G) Detection of global DNA methylation levels by DNA dot blot, (a) 5mC, (b) 5hmC. (H) Quantification of 5mC and 5hmC levels after withdrawal of decitabine. (I) Detection of Dnmt transcript levels by qRT-PCR. Fold changes are calculated relative to transcript levels at D2 or D8 control; n = 3 ± standard error of the mean. Significant differences from control: **P <0.01; *P <0.05. MB, methylene blue (used for visualizing total DNA).