Figure 2.

miR-449a suppresses neuronal cell cycle during differentiation.A, neurons derived from rat embryo cortex were seeded and allowed to differentiate for 2, 5, and 7 days in vitro (DIV). qRT-PCR was performed to detect miR-449a expression and fold change with respect to its expression on day 2 was determined. Data represent mean ± SEM, ∗∗p < 0.01, One-way ANOVA with Tukey’s post hoc test, N = 3. B, rat cortical neurons transfected with antagomir for miR-449a (B) or a control antagomir. After 48 h, lysates were prepared and subjected to immunoblotting with antibodies against PCNA, cyclin D1, and cl_caspase 3. Right panel, densitometry for indicated proteins was performed and fold change in the expression after normalization with respect to actin was determined. Data represent mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, two tailed paired t test, N = 3. C, rat cortical neurons were transfected with anti-miR-449a or a control inhibitor for 48 h followed by incubation with BrdU. Subsequently, immunofluorescence and TUNEL assays were performed to detect BrdU incorporation or apoptosis (green), respectively. Scale bar represents 100 μm (main image), 10 μm (inset). Right panel, % cells that were only stained with BrdU⁺, TUNEL⁺ (green), or both BrdU⁺/TUNEL⁺ (yellow) was determined (mean ± SEM, ∗p < 0.05, ns-not significant, two tailed paired t test, N = 3).