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. 2017 May 9;8(5):1256–1269. doi: 10.1016/j.stemcr.2017.04.006

Figure 4.

Figure 4

PI3K/AKT/mTOR Activity Promotes DNA Hydroxymethylation during Differentiation Induced by VPA

(A and B) The expression of TET1, TET2, and TET3 proteins was analyzed by immunoblotting and immunofluorescence staining. Scale bar, 250 μm. Representative graphs of three independent experiments.

(C) Dot blot analysis of 5-hmC quantity during differentiation of NSCs. Dot blot performed by biotinylation of 5-hmC followed by probing with Avidin-HRP. Dot blot representative of three independent experiments.

(D) Immunofluorescence staining for 5-hmC showed that the intensity of VPA on 5-hmC-immunostaining was attenuated by rapamycin (Rapa). Scale bar, 250 μm. ∗∗p = 0.001 < 0.01 compared with control; ##p = 0.005, 0.003 < 0.01 compared with VPA. Data are from three independent experiments.

(E) Relative quantitation of 5-hmC within a specific locus was performed using EpiMark 5-hmC Analysis Kit followed by real-time PCR analysis. ∗∗p = 0.002 < 0.01 compared with control; ##p = 0.004, 0.006 < 0.01 compared with VPA. One-way ANOVA and Student's t test were used to determine the statistical significance. Data are from four independent experiments.