Figure 2: 6XCBF-1 luciferase activity in WT and Nuc1 astrocytes transfected with Notch.

(a) In WT astrocytes transfected with the Notch-FL plasmid, luciferase activity in untreated astrocytes was significantly higher (4-fold increase) than in untreated vector-transfected astrocytes (*P=0.016). Significant induction of luciferase activity was observed in the presence of EDTA in WT astrocytes expressing Notch-FL plasmid (*P=0.031), while luciferase activity was significantly reduced in the presence of the γ-secretase inhibitor, DAPT (*P=0.009) compared with untreated astrocytes. Dimethylsulphoxide (DMSO; vehicle for DAPT) alone did not affect expression significantly. Luciferase activity in WT astrocytes expressing the NδE construct was not increased by EDTA, but existing activity was inhibited by DAPT (*P=0.012). Vector-transfected cells showed basal levels of luciferase activity. (b) In Nuc1 astrocytes, transfected with the Notch-FL plasmid, there was no significant induction of luciferase activity in the presence of EDTA. Luciferase activity was significantly reduced in the presence of DAPT (*P=0.025) compared with untreated cells. Luciferase activity in Nuc1 astrocytes expressing NδE was not induced in the presence of EDTA but could be inhibited in the presence of DAPT(*P=0.007). Vector-transfected cells showed basal levels of luciferase activity. (c,d) qRT–PCR analysis for expression of Hes1 and Hey1 in WT and Nuc1 astrocytes. Nuc1 astrocytes showed about 65 and 75% reduction in the levels of Hes1 and Hey1, respectively, compared with WT astrocytes (*P=0.030 and *P=0.027, respectively). In all panels, graphs show mean values and error bars represent s.d. from a triplicate experiment representative of at least three independent experiments. Statistical analysis was performed by a two-tailed Student’s t-test: *P<0.05.