FIGURE 3.

ERK5 signaling is necessary for promoting neurogenesis of SGZ-derived aNPCs in culture. aNPCs were infected with nonspecific shRNA control retroviral vector (shNS) or shRNA to ERK5 retrovirus as indicated. Both retroviral vectors encode eGFP marker protein under a bicistronic promoter. One day after virus infection, cells were washed and then incubated in culture medium free of EGF and bFGF for 5 days to allow spontaneous differentiation. Cells were then fixed for immunocytochemistry and co-stained for GFP to identify virus-infected cells (green) and various cell-type specific markers (red). A–D, co-staining of GFP with β-III tubulin. E, quantification of the percentage of GFP⁺ cells that are also β-III tubulin⁺. F–I, immunostaining of GFP⁺-infected cells and Nestin⁺ neural stem cells. J–M, immunostaining of GFP⁺-infected cells and PCNA⁺ proliferating cells. N, quantification of the percentage of GFP⁺ cells that also express Sox2, Nestin, or PCNA. Scale bar in A represents 25 μm and applies to all images. Arrowheads point to GFP⁺ cells co-labeled with various cell markers, whereas arrows point to GFP⁺ cells that are marker negative.