Figure 4.

Enforced Expression of miR-34b/c Promotes ESC DA Differentiation by Downregulating Wnt Signaling

(A) Schematic representation of the experimental procedure. mESCs were infected with an inducible lentiviral vector expressing miR-34b/c upstream of an Ires-GFP sequence. Cells were FACS purified, amplified, and differentiated toward the DA phenotype.

(B) TaqMan assay for miR-34c in FACS purified mESCs in presence or absence of doxycycline (DOX). Data were normalized to the average of the reference sno-202. Data represent mean ± SEM. ^∗p < 0.01 (Student's t test).

(C and D) qPCR analysis of genes related to the Wnt pathway (Wnt1, Lmx1b, Axin2, and Lef1; C) and dopaminergic lineage (Th, Vmat2, Dat, and Pitx3; D) at day 14 MD in the presence or absence of DOX. Data represent mean ± SEM from three independent experiments. ^∗p < 0.01 (Student's t test).

(E and F) Immunostaining and quantifications for TH in mESCs at day 14 MD (E). Counting was performed from 20 randomly selected fields for each condition, in three independent experiments. Data represent mean ± SEM. ^∗p < 0.05 relative to −DOX (Student's t test). A higher-magnification image of DA-differentiated ESCs is shown in (F). TH (red) and TUBB3 (green). Scale bars represent 200 μm in (E) and 100 μm in (F).