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. 2013 Mar 27;8(3):e60503. doi: 10.1371/journal.pone.0060503

Figure 3. DNA methylation marks and neuronal maturation in P7 dentate gyrus (DG) and effect of alcohol (see also quantitation in Figure 8).

Figure 3

Immunostaining of DNA methylation marks, 5mC (A–C) and 5hmC (D–F), 5hmC-converting enzyme TET1 (G–I), DNA methylation binding protein MeCp2 (J–L), and mature neuronal marker NeuN (M–O). There is a methylation gradation correlating with the Outside-In pattern of neuronal maturation in DG (M), in which the 5mC-im decreases as granule cells mature (A: DGi>DGo), while 5hmC-im increases as granule cells mature (D: DGi<DGo). Alcohol exposure derailed the reduction of 5mC in DGo (C, star) and the acquisition of 5hmC in both DGi and DGo (F, star), which is accompanied by the delayed maturation of DG as indicated by decreasing the NeuN+ cells (O, star). There is no significant difference between PF and Chow. The TET1 protein expression is closely associated with the expression of 5hmC (DGi<DGo), which was also reduced by Alc (I, see star). The expression of MeCP2 known to correlate with neuronal maturation was also reduced by alcohol (L, star). DGo: dentate gyrus outer layer; DGi: dentate gyrus inner layer. Scale bar: all = 100 µm.