To further study the impact of DNA demethylation/remethylation in Mecp2 isoform-specific expression, we treated dissociated neurosphere cells with 2.5 μM decitabine for 48 h, at the onset of NSC differentiation (D0) (Figure 4A). At D2, decitabine was withdrawn from the media and cells were kept in culture for another 6 days until D8, to study the effect of DNA remethylation (Figure 4A). First, as a proof of principle, we verified whether decitabine acted as a DNA demethylating agent in our system. Global change in DNA methylation was determined by IF for 5mC and DNA dot blot assay for both 5mC and 5hmC. As expected, IF experiments showed that 5mC nuclear signals were noticeably lower in decitabine-treated NSC compared to D2 control untreated cells (Figure 4B). DNA dot blot assays indicated that decitabine treatment resulted in reduced 5mC levels (3.79-fold, P <0.05), with slight but statistically insignificant increase in 5hmC levels (Figure 4C-D). In contrast, decitabine withdrawal led to re-establishment of global DNA methylation (5mC) at D8 as detected by IF (Figure 4F). Furthermore, DNA dot blot assay showed DNA methylation
