Fig. 2
PINK1 deficiency did not affect the self-renewal or proliferation capacities of NSCs. a, b The secondary neurosphere formation ability was assayed in WT and PINK1-KO NSCs. Dissociated primary neurospheres (2 × 104 cell/well) were seeded to a 96-well plate in the presence of EGF (20 ng/ml) and FGF (20 ng/ml). On day 3 of seeding, the sizes and numbers of secondary neurospheres were analyzed. c, d NSC proliferation was determined by counting of cell numbers (c) and measurement of [3H]-thymidine incorporation (d). Dissociated NSCs were plated to 24-well plates coated with poly-L-ornithine and fibronectin (1 × 105 cell/well) in the presence of EGF and FGF. On the following day, 1 μCi/ml [3H]-thymidine was added with growth factors, and the plates were incubated for an additional 24 h. Finally, the NSCs were washed with PBS and lysed with 0.1 N NaOH, and radioactivity was determined using a β-counter. e WT and PINK1-KO NSCs were subcultured every 3-4 days after neurosphere formation in the presence of 20 ng/ml EGF and FGF until passage 8. At each passage, the number of cells was counted. Values in (b, c) are means ± SEMs of at least three samples. Scale bar in (a), 200 μm. The data shown are representative of at least three independent experiments