Figure 4

MYC-dependent regulation of NOTCH ligand JAG2 expression. (a) Relative mRNA expression (upper panel) of MYC (black bars, left Y axis) and JAG2 (orange bars, right Y axis) in the indicated cell lines. Values represent the fold-change in mRNA expression relative to cerebellum (defined as 1). Protein expression (lower panel) of MYC and JAG2 in the corresponding cell lines. (b) Relative mRNA expression (upper panel) of MYC and JAG2 in DAOY cells and stable clones of DAOY V11, and DAOY M2.1 cells. Values represent fold-change in mRNA expression relative to cerebellum (defined as 1). Protein expression (lower panel) of MYC and JAG2 in corresponding cells is shown. (c) MYC and JAG2 mRNA (upper panel) and protein (lower panel) expression in DAOY M2.1 cells transfected with MYC siRNA compared to control siRNA. mRNA values represent the percent decrease in JAG2 and MYC expression relative to control. Expression of β-actin was used as a control for western blot analysis. Statistical analysis: black stars indicate p values relative to MYC expression; orange stars indicate p values relative to JAG2 expression. (d) ChIP-on-chip data show occupancy of the JAG2 genomic sequence by MYC in four MB cell lines (from top to bottom: DAOY, UW-228, D458, and D341). (e) Time-dependent caspase 3/7 activation in DAOY M2.1 cells upon JAG2 siRNA treatment at the indicated time-points; values represent the percent increase in caspase 3/7 activity relative to control. (f) Proliferation status of DAOY cells, DAOY V11, DAOY M2.1, and DAOY M2.1 cells at 48 hours after JAG2 siRNA treatment compared to control siRNA. Caspase 3/7 activation at 48 hours after treatment with GSI in the indicated cell lines (g) and DAOY-derived clones (h). Low MYC cells: DAOY; high MYC cells: DAOY M2.1. Values represent the percent increase in caspase 3/7 activity relative to control siRNA.