Expression of IAP2 mRNA in NSPCs.

<p>(A) Differential display analysis of 13 DIV neurosphere (left lane) and 20 DIV neurosphere (right lane) RNA using No. 22 upstream primer and 5′-T_nAC-3′ downstream primer sets. Arrowhead indicates the differentially expressed IAP2 cDNA fragment between 13 DIV neurospheres and 20 DIV neurospheres. (B) Schematic representation of the alternative splicing possibilities for generation of IAP forms. Alternative splice forms of IAP were identified by RT-PCR analysis using primer pairs designed in common exon sequences (arrows). Right arrow indicates the position of the forward primer and the left arrow indicates the position of the reverse primer for RT-PCR as shown in (C). (C) Identification of splicing forms of IAP expressed in NSPCs using RT-PCR analysis. Total RNA was isolated from 13 DIV neurospheres and 20 DIV neurospheres. Alternative splice forms were distinguished by the size of PCR products using primer sets shown in (B). (D) Quantification of IAP2 mRNA expression levels in 13 DIV neurospheres and 20 DIV neurospheres. Total RNA was isolated from 13 and 20 DIV neurospheres. The expression levels of IAP2 and Ppia (internal standard) mRNA were determined by real-time quantitative PCR analysis. (E) Western blot analysis of IAP protein expression at 13 DIV (left lane) and 20 DIV (right lane). Each value represents the mean ± SEM from three independent experiments and is expressed in reference to 13 DIV neurospheres. **<i>p</i> < 0.01 compared with 13 DIV neurospheres.</p

<i>In vitro</i> time-dependent decline in neurogenic competency of NSPCs.

<p><b>(A)</b> Strategy for the functional screening of candidate genes involved in the temporal specification of NSPCs using the neurosphere method. (B) Representative immunofluorescent images of NSPCs differentiated from 13 DIV neurospheres (left) and 20 DIV neurospheres (right). Secondary neurospheres were dissociated in NSPC proliferation medium and plated on poly l-lysine-coated cover glass. After 24 hours, NSPCs were exposed to NSPC differentiation medium, fixed at 10 days after differentiation, stained for a neuron marker (Tuj1; green) and an astrocyte marker (GFAP; red), and counterstained with DAPI (blue). Scale bar: 50 μm. Quantification of Tuj1-positive cells (C) or GFAP-positive cells (D) 10 days after differentiation. Data are shown as the mean ± SEM of four independent experiments. *<i>p</i> < 0.05.</p