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. 2016 Sep 29;7:12733. doi: 10.1038/ncomms12733

Figure 7. FoxO1 directly regulates tyrosine hydroxylase (TH) expression in DA neurons.

Figure 7

(a) mRNA level of Th in DA neurons of WT and KO littermates. (b) Immunoblots for TH and phosphorylated TH (p-TH) in SN and midbrain from WT and KO littermates. (c) Relative TH and p-TH protein levels from b. (d) Top, schematic diagram for mouse TH promoter. Bottom, ChIP assays using whole-brain and Neuro2A cells transfected with myc-tagged FoxO1-ADA showing a direct and specific binding of FoxO1 on the proximal region of TH promoter. (e) Top, schematic diagram for luciferase constructs with or without FoxO1 potential binding sites. Bottom left, relative luciferase activity after FoxO1-WT (WT), constitutive active form of FoxO1 (ADA) and dominant-negative form of FoxO1 (DN) overexpression (n=6). Bottom right, immunoblots confirming expression of FoxO1-WT, -ADA, and -DN. (f) Th mRNA expression in Neuro2A cells after FoxO1-WT and -ADA overexpression (n=9). (g) Th mRNA level in Neuro2A cells after FoxO1 knockdown (n=9). The results are expressed as mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001, Student's t-test and one-way analysis of variance for luciferase and quantitative real-time PCR analyses) from more than three independent experiments.