Status of SNCA methylation in SK-N-SH cells and impact on expression. a, SK-N-SH cells were incubated with DMSO [control (Ctrl)] or 10 μm Aza for 24 and 48 h, respectively, and isolated DNA was used for bisulfite sequencing of SNCA(−2079/−1507) (promoter) and SNCA(−926/−483) (intron); 10 independent clones for each condition were analyzed by bisulfite sequencing. The lollipop diagram with unmethylated CpG sites (open circles) and methylated CpG sites (closed circles) illustrates the higher degree of methylation in SNCA(−926/−483) (intron) compared with SNCA(−2079/−1507) (promoter) and the decrease of methylated CpG in SNCA(−926/−483) (intron) after addition of Aza. b, After 48 h of Aza treatment, the fraction of methylated CpGs is reduced by ∼60% in SNCA(−926/−483) (intron). c, Treatment with Aza induced a time-dependent increase of SNCA expression exceeding 50% of Ctrl. Quantitative RT-PCR was performed in triplicate and presented as the mean ± SD. d, Representative Western blot with anti-synuclein antibody demonstrates an increase of synuclein protein after addition of Aza too.