Figure 6.

A, Immunocytochemistry for the neuronal marker Tuj1 (green) and nuclear stain Hoechst (blue) of 1 μm H₂O₂ preprimed wild-type (wt) and p53−/− differentiating NPCs (DIV 5). Neurospheres were pretreated with H₂O₂ in proliferation conditions and plated subsequently for differentiation. B, The bar graph shows that pretreatment of neurospheres with H₂O₂ increases neurogenic differentiation in wild-type but not in p53-null NPCs. Data represent the mean percentage of Tuj1+ cells normalized to the total number of differentiating cells (Hoechst+ cells) ± SEM from three independent experiments. C, Immunocytochemistry for the neuronal marker Tuj1 (green) and nuclear stain Hoechst (blue) of antioxidant N-acetyl-cysteine (1 mm) pretreated wild-type and p53−/− differentiating NPCs (DIV 5). Neurospheres were pretreated with NAC in proliferation conditions and plated subsequently for differentiation. D, The bar graph shows that pretreatment of proliferative NPCs with antioxidant NAC decreases neurogenic differentiation in p53−/− cells only. Data represent the mean percentage of Tuj1+ cells normalized to the total number of differentiating cells (Hoechst+ cells) ± SEM from three independent experiments. ^★★p < 0.01; ^★★★p < 0.001 (two-tail t test). E, Immunoblotting of E16 p53−/− telencephalic lysates for neuroblast/immature neuron marker DCX, whose expression is reduced by NAC. Pregnant dams were injected daily from E10 onward, either with control saline or antioxidant NAC. Embryos were killed at E16. Antioxidant treatment decreased the amount of DCX (N = 3). Scale bars: 100 μm.