Figure 3: Effect of Jagged1 on Notch signalling in WT and Nuc1 astrocytes.
From: Impaired endolysosomal function disrupts Notch signalling in optic nerve astrocytes
(a) Western analysis for Jagged1 in WT and Nuc1 astrocytes transfected with scrambled or ON-TARGET plus Smart pool Jagged1 siRNA. Downregulation of Jagged1 expression was observed in both WT and Nuc1 astrocytes transfected with the targeted siRNA. (b) qRT–PCR for the Notch pathway targets Hes1 and Hey1 in WT and Nuc1 astrocytes after treatment with scrambled or Jagged1 siRNA. Knockdown of Jagged1 expression (*P=0.013 in WT and *P=0.017 in Nuc1) is reflected by decreased expression of Hes1 (*P=0.022 in WT and *P=0.015 in Nuc1) and Hey1 (right panel: values relative to scrambled siRNA in left panel). (c) 12XCSL-luciferase reporter activity in cocultures of Notch1-expressing astrocytes carrying 12XCSL-luciferase reporter construct (ΔEGF) or pRL-cmv control (EGF) construct, with astrocytes overexpressing the Jagged1 ligand. Graph shows luciferase activity (arithmetic mean) from triplicates of each coculture. Robust luciferase activity was seen in WT astrocytes (*P=0.011), compared with Nuc1 astrocytes. This increase is compromised in astrocytes expressing the Jagged1 ΔEGF Notch-binding-deficient plasmid. (d) 12XCSL-luciferase reporter activity in cocultures of Jagged1 knockdown astrocytes expressing 12XCSL-luciferase reporter construct or pRL-cmv control constructs with astrocytes overexpressing Jagged1. A marked induction of Notch signalling was observed in both WT (*P=0.019) and Nuc1 astrocytes transfected with Jagged1 siRNA (*P=0.027) and cocultured with astrocytes overexpressing Jagged1 compared with scrambled siRNA-treated cells (left panel). Astrocytes transfected with empty vector were also cocultured with Jagged1 overexpressing cells, showing a modest induction of the Notch pathway in WT and none in Nuc1 homozygous astrocytes. 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.
