Fig. 3.

The effect of the extended α-SYN 3′UTR on α-SYN translation and their level changes in iPSC-derived dopaminergic neurons. a Firefly luciferase reporter constructs containing the annotated (2.5 kb) or extended (3.8 kb) form of α-SYN 3′UTR. b Luciferase activity from SH-SY5Y cells co-transfected with firefly luciferase containing either 2.5 or 3.8 kb α-SYN 3′UTR and Renilla luciferase. The firefly luciferase values were normalized to Renilla luciferase activity. c RT-PCR for total α-SYN transcripts, the extended α-SYN 3′UTR and β-actin from iPSC-derived dopaminergic neurons (DIV 60). RNA samples from total 12 iPSC lines; three iPSC clones from each patient (two control; CTRL1 and 2, two sporadic PD; sPD1 and 2), were used. β-actin was used as an internal control. “+” or “-­” RT; with or without RT reaction. d Quantitative analysis of total α-SYN mRNA expression after normalization by β-actin. e Quantitative analysis of the extended α-SYN 3′UTR expression after normalization by β-actin. f Western blotting for α-SYN, tyrosine hydroxylase (TH), and β-actin from iPSC-derived dopaminergic neurons (DIV 60). β-actin was used as an internal control. g Quantitative analysis of α-SYN protein expression after normalization by β-actin. h Quantitative analysis of TH protein expression after normalization by β-actin. i Reverse correlation between the extended α-SYN 3′UTR and α-SYN protein levels. The Pearson’s correlation coefficient = − 0.6688. Error bars denote mean ± S.E.M. n.s (not significant), **P < 0.01, ***P < 0.001, ****P < 0.0001 by unpaired two-tailed t test in d, e, g, h