Fig. 2. Myc overexpression increases proliferation and self-renewal in NPCs. (A) Overexpression of Myc increases proliferation as shown by the proportion of the BrdU-positive NPCs in neurosphere cultures. During 24 hours of exposure to BrdU, 28.9% of the control NPCs (s.d.=5.6, n=4) were in cell cycle whereas Myc overexpression increased the rate to 76.7% (s.d.=5.3, n=5, Student's t-test **P=7.2E-06). (B) Myc increased the total cell number 70% in 1 week (from 3448, s.d.=274, n=3 to 5810, s.d.=1004, n=3, Student's t-test **P=0.00149). (C) Self-renewal assay set up and result in which Myc increases the proportion of self-renewing NPCs by fivefold from 4.2% (s.d.=2.2, n=13) in controls to 24.0% (s.d.=8.7, n=10, Student's t-test **P=0.000104) in the Myc-overexpressing neurospheres. The ability of single cells to form new neurospheres was measured by microscopy after 7 days of culture in the EGF- and FGF-containing stem cell medium. (D) Flow cytometry shows quantitatively that Myc overexpression increases the expression of the neural progenitor markers nestin (74.3%, s.d.=11.3, n=10) and Bmi-1 (93.3%, s.d.=4.1, n=11) as compared with the control NPCs (nestin 44.2%, s.d.=13.9, n=5 and Bmi-1 84.5%, s.d.=6.2, n=7, Student's t-test for nestin **P=0.0043 and for Bmi-1 **P=0.0085). (E) Even if the self-renewing proportion of the heterogenous neurosphere cell population is increased, Myc overexpression does not affect the expression of differentiation markers GFAP and TUJ-1 and thus the proportion of differentiated cells in the neurosphere (control TUJ-1 3.7%, s.d.=4.3, n=6; GFAP 15.2%, s.d.=7.9, n=6 and Myc TUJ-1 8.7%, s.d.=4.5, n=10; GFAP 15.8%, s.d.=12, n=9).