Fig. 3
PINK1 expression increased during differentiation of NSCs, and its deficiency caused defects in GFAP-positive astrocyte differentiation. NSCs (1.5 × 106 cell/well) were prepared from E13.5 WT and PINK1-KO mouse embryo brain and seeded to 6-well plates coated with poly-L-ornithine/fibronectin. Differentiation was induced by withdrawal of EGF and FGF. a, b At the indicated times after induction of differentiation, protein levels of Nestin, MAP2, TUJ-1, and GFAP (a), and protein and mRNA levels of PINK1 (b) were assayed, and the results were quantified (b). c The differentiation capacities of WT and PINK1-KO NSCs into astrocytes (GFAP) and neurons (TUJ-1 and/or MAP2) were analyzed by Western blot (left panel) and quantified (right panel). d The differentiation capacities of WT and PINK1 KO NSCs into astrocytes (GFAP) were analyzed in the presence of CNTF by Western blot (left panel) and quantified (right panel) on day 5 of differentiation. GAPDH or actin was used as the loading control. e On day 5 of differentiation, cells were immunostained for cell-type-specific markers, neuron (MAP2), astrocyte (GFAP), and oligodendrocytes (CNPase) (left panel). Images were taken using a Zeiss microscope, and the number of each type of cells was counted using the Image J software, and plotted (right panel). Scale bar in (e), 50 μm. Values in (b, c, d, and e) are means ± SEMs of at least three samples (**, P < 0.01). The data shown are representative of at least three independent experiments