Figure 5

Effect of decitabine exposure and withdrawal on Methyl CpG binding protein gene ( Mecp2 /MeCP2) expression. (A-C), After exposure to decitabine at D2. (A) Analysis of Mecp2 (total), Mecp2e1 and Mecp2e2 transcript levels by qRT-PCR. (B) Detection of MeCP2 (total) protein expression levels by western blot; n = 2 ± standard error of the mean (SEM). (C) Detection of MeCP2E1 protein expression levels by western blot; n = 2 ± SEM. (D-F), After withdrawal of decitabine at day 8 (D8). (D) Analysis of total Mecp2, Mecp2e1 and Mecp2e2 transcript levels by qRT-PCR. (E) Detection of MeCP2 (total) protein expression levels by western blot. (F) Detection of MeCP2E1 protein expression levels by western blot. (G-H) Pearson's correlation analysis of the relation between Mecp2 transcript levels and MeCP2 protein levels at D2 (G) and D8 (H); r = Pearson’s correlation coefficient, r² = coefficient of determination. (I) Transcript detection of cell type-specific markers for neurons (Tub III, NeuN); astrocytes (Gfap, S100b); oligodendrocytes (Cnpase, Mbp) by qRT-PCR in D2 control and decitabine-treated cells. (J) Quantification of neurons, astrocytes and oligodendrocytes using cell type-specific markers by immunofluorescence in D2 control and decitabine-treated cells. (K) Transcript detection of cell type-specific markers for neurons (Tub III, NeuN); astrocytes (Gfap, S100b); oligodendrocytes (Cnpase, Mbp) by qRT-PCR in D8 control and decitabine-treated cells. (L) Quantification of neurons, astrocytes and oligodendrocytes using cell type-specific markers in D8 control and decitabine-treated cells. (M) Comparison of immunofluorescent detection of (a) Glial fibrillary acidic protein (GFAP) and (b) S100B between control and decitabine-treated cells. Images were taken at the same exposure time. Scale bars represent 20 μm. For all the panels, fold changes are calculated relative to expression levels at D2 or D8 controls. Significant differences from controls: ^****P <0.0001; ^***P <0.001; ^**P <0.01; ^*P <0.05, n = 3 ± SEM, unless specifically mentioned.