Figure 2.

miR-218 upregulation positively affects dopaminergic differentiation. a, Schematic representation of the experimental procedure adopted for cell-based High Content Screening. b, Representation of the genomic sequences encompassing miRNAs genomic loci cloned into the TET-O-FUW plasmid for the production of inducible lentiviral particles (LVs) used to overexpress miRNAs. c, Representative images of E12.5 midbrain primary cultures infected with LVs for Nurr1 alone or in combination with Lmx1a. Immunostaining was performed against 3xFlag-NURR1 (red) and Th (green), nuclei were stained with DAPI (blue). Images were acquired at 20× magnification. Scale bar, 50 μm. d, Relative ratio of TH⁺/NURR1⁺ cells following lentiviral infection with 3xFlag-Nurr1 alone or in combination with LVs for different miRNAs. Cells infected with Nurr1 alone were used as basal control (=1). Data are mean ± SEM. n = 270 ± 80 acquired fields from at least 5 independent experiments. *p ≤ 0.05 (one-way ANOVA + Benjamini-Hochberg's post hoc test). e, Representative images of E12.5 midbrain primary cultures (mE12.5-PCs) overexpressing by LVs Nurr1 and/or miR-218-1 and differentiated for 12 d. Cells were stained for 3xFlag (exogenous NURR1, red), TH (green), and DAPI (blu). Images were acquired at 20× magnification of a Leica DMI600 microscope. Scale bar, 50 µm. f, g, Automatic counts of TH⁺ (f) and total (g) cells from mE12.5-PCs overexpressing single miRNAs or Nurr1 (3x-Flag). Total cells were identified with the nuclear dye (DAPI), while TH⁺ cells were stained for TH as control cells infected with the only rtTA were used. Dashed lines indicate the mean for Ctrl values.