Figure 3

Overexpression of miR-124 in vitro and in vivo. (A) Backbone of the expression plasmids pPri-miR-124(2) and pPri-miR-155/neg_control; miR-124-responsive sensor plasmid pCMV-DsRed2/Lhx2 _3'UTR. The asterisk indicates the position of Pri-miR cDNA fragments. (B, C) Specific attenuation of DsRed2 expression in HeLa cells cotransfected with pPri-miR-124(2) and pCMV-DsRed2/Lhx2 _3'UTR. PT, post-transfection. (D) Backbone of lentivectors LV_Pri-miR-124(2) and LV_Pri-miR-155/neg_control. The asterisk indicates the position of Pri-miR cDNA fragments. (E) DsRed2 expression in E12.5 primary cortico-cerebral progenitors infected by lentiviruses LV_Pri-miR-124(2) and LV_Pri-miR-155/neg_control at a multiplicity of infection of 40 and kept for 30 h in DMEM:F12:N2 medium supplemented with 2.5% fetal calf serum. PI, post-infection. BF, bright field. (F, G) Differential β-tubulin immunoprofiling of acutely infected, E12.5 dissociated cortical progenitor cells, at 72 h after infection. (H) Example of neurite outgrowth evaluation by immunostaining and subsequent NeuriteTracer^® analysis. (I) Differential neurite outgrowth in low density cortical progenitor cells 72 h after infection at E12.5, calculated for three different experiments by NeuriteTracer^®. (J, K) In vivo overexpression of miR-124 in lateral neocortex. Distribution of miR-124 and pCMV-driven EmGFP on E14.5 midfrontal telencephalic sections from brains electroporated at E12.5 with the plasmids pPri-miR-124(2) and pPri-miR-155/neg_control, respectively. Magnifications of boxed insets of electronic merges are shown to the right. Abbreviations: bg, basal ganglia. In (C) error bars represent the standard error of the mean calculated among the means of each experiment; **P < 0.01, as evaluated by ANOVA test; N = 3+3. In (G, I) error bars represent the standard error of the mean calculated within each experiment; the P < 0.01 value was evaluated by t-test (one-tail, paired); N = 3+3. Scale bars = 40 μm (H) and 100 μm (I).