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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Stem Cells. 2015 Apr;33(4):1320–1332. doi: 10.1002/stem.1932

Figure 4. VC reduces the repressive histone mark H3K27m3 on chromatins of dopamine (DA) phenotype genes through Jmjd3.

Figure 4

(A): Specific reduction of H3K27m3 but not other modifications by VC is shown as a representative Western blot. Histone 3 (H3) expressions were used as the loading control and the reference for quantification of the histone modifications in the blot analyses. (B): Protein expression pattern of H3K27m3 along differentiation. A representative Western blot is shown on the left. Quantification of intensity is shown on the right. (C): Colorimetric measurement showing nuclear-specific increase of Jmjd3 enzyme activity by VC with cytosolic or nuclear extracts from VC treated or non-treated samples. (D): ChIP-qPCR analysis of various histone modifications on Th promoter region from samples treated with or without VC. (E): Enrichments of H3K27m3 on promoters of DA phenotype genes are specifically decreased in the DA neuron-specific gene promoters, along with no significant change of those on promoters specific for serotonergic (Tph2) and GABAergic (Gad67) neurons. Data are presented relative to no VC treatment. (F, G): Pretreatment of the H3K27m3 inhibitor GSK-J4 abolishes VC-induced demethylation of H3K27m3 (F) and TH+ cells increase (G). VC (10 µM) was present in vehicle and GSK-J4 treated cultures. (H): qRT-PCR analysis for expression of DA phenotype genes altered by GSK-J4 pretreatment. (I): Decreased enrichments of Nurr1 on promoters of DA phenotype genes by GSK-J4 treatment. Abbreviations: TH, tyrosine hydroxylase; VC, vitamin C. *, p < .05 and **, p < .01, one-way ANOVA.