Figure 3

Blockade of Notch signaling promotes the differentiation of normal mouse NSCs into INPs and downstream neural cell types. (A, B) Total RNA was prepared from GSI or DMSO treated neurospheres derived from E12.5 mouse brain on the 5th day of culture. And the expressions of Glast, Mash1 and Tubulin α1 were measured by RT-PCR (A) and Real-time PCR (B), with β-actin as the reference control (n = 3, GLAST, P = 0.003, Mash1, P = 0.043, Tubulin α1, P = 0.046). (C, D) Immunofluorescence. Differentiated NSCs were stained with anti-GFAP, or anti-MAP2 antibodies after cultured on cover slips in differentiation conditional medium for 7 days. Stained samples were examined under a fluorescence microscope. (E, F) Quantification and comparison of neurons (MAP2⁺) or astrocytes (GFAP⁺) in GSI-treated and control NSCs. Cells were counterstained with Hoechst, to permit counting of cell nuclei in at least 5 microscopic fields per specimen (n = 3, E, P = 0.021, F, P = 0.031). *, P < 0.05, **, P < 0.01. Scale bar, 50 μm for C and D.