Figure 2.
Temporal expression of various differentiation markers and of RTP801 in plated neurosphere cultures. A, Time course of neural stem cell differentiation after plating on an adherent substrate. After disaggregation and plating, murine hippocampal neurosphere cultures were stained at the indicated times with specific antibodies to detect undifferentiated neuroprogenitor/neural stem cells (nestin), neurons (β-III tubulin), astrocytes (GFAP), and oligodendrocytes (O1). Proliferating cells were also visualized by incubating with and immunostaining for bromodeoxyuridine (BrdU). B, RTP801 is transiently induced in plated neurosphere cultures. Extracts from floating neurospheres (NS), disaggregated neurospheres (dNS) (as a control for mechanical stress), and neurospheres that were disaggregated and plated for the indicated times were analyzed by Western immunoblotting for RTP801 expression. Membrane was reprobed against Erk1/2 as a loading control. C, RTP801 is present at high levels in neuroprogenitor cells and newborn neurons, but not in astroglia or mature neurons in plated neurosphere cultures. Plated neurospheres cultures were subjected to fluorescence immunostaining for RTP801 and the indicated markers 1 and 8 d after disaggregation and plating. Hoechst dye 33242 was used to stain nuclei. Scale bars: top, 10 μm; bottom, 20 μm.