Figure 3.

Qki is expressed in neural progenitor cells and targeted by miR-214. (A) Screening with 3′UTR luciferase reporter assays to determine functional miR-214 binding sites. Bar graphs show the relative luciferase activity of 26 potential target gene 3′-UTRs in HEK-293ET cells after overexpression of miR-214. Data were normalized to Renilla luciferase activity. (B) The mRNA expression of Fezf1, Ezh1, Qki and Ppme1 in the mouse telencephalon at E12.5, performed by in situ hybridization. (C) The dynamics of Qki mRNA expression during cerebral cortex development at E12.5, E14.5, E16.5 and E18.5. (D,E) Western blot analysis of QKI expression after overexpression of miR-214 for 48 hours in N1E-115 cells (D) and HEK-293ET cells (E). β-actin was included as a loading control. Uncropped versions of all western blots are shown in Supplementary Figure 3. (F) The structures of the three major alternative splice variants of Qki and the predicted miR-214 binding sites in their 3′ UTRs. The “−” sign over the black line represents the possible miR-214 binding sites. (G) Dual luciferase assays of 293ET cells co-transfected firefly luciferase construct containing the wild-type(WT) or respective target sites mutant(Mut) 3′-UTR of the Qki isforms, along with the miR-214 expression plasmid or the empty vector. All experiments were normalized to co-transfected Renilla luciferase. Histograms show normalized mean values of the relative luciferase activity, from three or more independent transfections. Error bars show the standard deviation, and the comparisons were performed by Student’s t-test; the statistically significant P values are shown as *(<0.05), **(<0.01) or ***(<0.001). (H) The in situ hybridization of the three Qki variants with specific probes respectively shows their expression pattern in the cerebral cortex at E12.5 to E18.5. Scale bar: 100 μm.