Figure 3.

IGF2 regulates proliferation of DG NSCs in vitro. A, ELISAs for IGF2 were performed. Medium from proliferating DG and SVZ NSCs and from 14 d differentiated DG NSCs was collected, concentrated and analyzed. Graph shows the amount of Igf2 in the medium in pg/ml normalized to total protein (mg/ml). B, IGF2 protein levels are higher in DG-derived NSCs than in SVZ-derived NSCs. Total protein was isolated from proliferating NSCs isolated from DG or SVZ. Western blot analysis showed higher protein levels of IGF2 in DG- than in SVZ-derived cultures. C, IGF2 is downregulated with differentiation of DG-derived NSCs. Shown is a representative Western blot and quantification of relative protein levels. D, DG cells were infected with lentiviruses expressing different shRNA sequences targeting Igf2 mRNA. shRNA sequences sh1 and sh2 resulted in ∼75% and 60% decreases in IGF2 protein levels, respectively. Shown are representative Western blots with nontargeting shRNA (CON) or shRNA against Igf2. E, Knockdown of IGF2 using two independent lentiviruses expressing shRNAs (Igf2-sh1 and Igf2-sh2) targeting the murine Igf2 mRNA resulted in a robust decrease in the number of proliferating, EdU-labeled, DG-derived NSCs in vitro (top, red). Exogenous IGF2 rescued the proliferation defect caused by knockdown of endogenous IGF2 knockdown. IGF2 supplementation (20 ng/ml IGF2) rescued the proliferation phenotype (as measured by EdU incorporation, bottom, red) of IGF2 knockdown in DG-derived NSCs. Images show representative pictures of cells transduced with a lentivirus expressing nontargeting shRNA (CON, green, left) or expressing shRNA sequence 1 (Igf2-sh1) or 2 (Igf2-sh2). Arrowheads point to GFP-expressing, EdU-labeled cells. Data are presented as mean ± SEM. Scale bar, 50 μm. F, After transduction with viruses expressing shRNAs directed against Igf2 or nontargeting control shRNA, NSCs were differentiated for 14 d. There was no effect on the number of MAP2ab/GFP+ cells compared with control. Shown are examples and the quantification of GFP (virus-expressing, green) and MAP2ab (red) colabeled cells in DG NSC cultures. Nuclei were counterstained with DAPI (blue). Data are presented as mean ± SEM. Scale bar, 100 μm. G, IGF2 knockdown did not affect the number of EdU-labeled cells in NSC cultures derived from the adult SVZ. Right, representative pictures of cells transduced with a nontargeting shRNA expressing lentivirus (CON, green, left) or expressing shRNA sequence 1 (Igf2-sh1) or 2 (Igf2-sh2) (green, middle and right). Arrowheads point toward GFP-expressing, EdU-labeled cells. Scale bar, 50 μm. *p < 0.05.