Fig. 6.
Neuronal differentiation of NSC after pretreatment (8 h) with conditioned media or media from irradiated U87MG cells. a, b NSC at the initial density (3 × 105 cells/well) were used for pretreatment with transferred media and induction of differentiation. U87MG cells were non-treated or directly γ-irradiated at 5–10 Gy. Naïve NSC was primed by 8-h incubation with transferred media from non-irradiated glioblastoma cells (GB-0 Gy) or from irradiated glioblastoma cells (GB-5 Gy; GB-10 Gy). Fresh GB media was used as a control. Eight hours after GB media transfer, neuronal differentiation was initiated by replacement GB media with serum-free differentiation medium. Confocal analysis of immunofluorescent images was performed using monoclonal Ab against an early neuroprogenitor marker, Nestin (red), and polyclonal Ab against a neuronal marker, Doublecortin (green). Bar = 50 μm. A relative cell survival and a ratio of the number of green cells to the number of red cells (reflecting neuronal differentiation) 10 days after initiation of differentiation are indicated in b. c, d NSC at the higher initial cell density (1 × 106 cells/well) were used for pretreatment with transferred media and induction of differentiation. Pooled results of four independent experiments are shown in b, d. Error bars represent means ± SD (p < 0.05, Student’s t test); star indicates a significant difference