Effect of FGF21 on the expression and activation of PGC-1α in human dopaminergic neuron. Human dopaminergic neurons differentiated from midbrain progenitor cells were cultivated as described in Materials and methods. Cells were treated with 50 ng/ml FGF21 for 24 h as indicated below. (A) Left, immunoblot showing expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the human dopaminergic cells. β-actin was used as control. Right, phase contrast pictures. There was no significant difference in morphology between control and FGF21 treated cells. Scale bar. (B) Left, immunoblot. β-actin was used as control. The levels of PGC-1α were increased by FGF21. Right, quantification was done using ImageJ software. Values are means ± SD, n = 4, ***p < 0.001 for FGF21 vs C. (C) Gene promoter assays. Cells were transfected with the pGL3 basic plasmid and the PGC-1α promoter plasmids linked to a luciferase reporter. Cells were stimulated with FGF21 for 24 h and the luciferase activity was measured and corrected for that of Renilla as described as described in Methods. FGF21 enhanced PGC-1α gene activity but not that of the control pGL3 promoter. Values are means ± SD, n = 4, *p < 0.05 for FGF21 vs controls. (D) Immunoprecipitation experiments. PGC-1α was immunoprecipitated from control and FGF21-treated cells followed by immunoblotting as described in Methods. The degree of acetylation of PGC-1α was analyzed using the anti-acetylated lysine antibody. Total amount of PGC-1α in the immunoprecipitate was analyzed using anti- PGC-1α antibodies. Values are means ± SD, n = 4, *p < 0.05 for FGF21 vs C.