Blockade of Notch signaling attenuates proliferation and self-renewal ability of normal mouse NSCs. (A) Photomicrographs of neurospheres derived from E12.5 mouse brain at 72 h after primary culture, with GSI or DMSO supplemented. (B, C) Total RNA was prepared from neurospheres on the 5th day of GSI or DMSO treatment. And the expressions of Hes1 and Hes5 were measured by RT-PCR (B) and Real-time PCR (C), with β-actin as the reference control (n = 3, Hes5, P = 0.006, Hes1, P = 0.006). (D, E) Equal number of cells (1 × 105/ml) were plated in the growth medium, and the number of primary (n = 3, P = 0.010) (D) and secondary (n = 3, P = 0.043) (E) neurospheres were counted 7 days after plating. *, P < 0.05, **, P < 0.01.