Figure 1

(a) Western blot analysis for expression of activated Notch in transduced EC. Control EC (transduced with the empty LZRS retroviral vector) expressed transmembrane Notch-1 and -2 (TM, arrowheads, approximately 120 kDa) as well as activated N^IC-4 (N^IC arrow, approximately 75 kDa). In contrast, EC transduced with LZRS-N^IC-1 expressed both transmembrane and activated Notch-1 (N^IC arrow, 90–110 kDa), but did not express activated Notch-2, which confirmed the specificity of N^IC-1 overexpression. Similarly, cells overexpressing N^IC-2 possessed both the transmembrane and activated forms of Notch-2, but Notch-1 activation was not detected. As cultured EC constitutively express N^IC-4, cells transduced with LZRS-N^IC-4 demonstrated an increase in its expression (50% increase on average) with little to no effect on Notch-1 or Notch-2 expression. The results are representative of at least two independent experiments. (b) Induction of Notch transcriptional activity by overexpression of activated Notch. Luciferase activity was measured in EC transduced to express activated Notch and then transfected with a Hey-1-luc reporter construct. The results show significantly higher luciferase activity in N^IC-1, N^IC-2, and N^IC-4-transduced cells compared to vector control cells (3.3-fold, 4.0-fold, and 3.4-fold increase, respectively; P<0.01). Luciferase values were normalized for transfection efficiency. The results are combined data from two experiments performed in triplicate. (c) Notch activation inhibits EC proliferation. Using a WST-1 assay, we found a significant decrease in proliferation of EC overexpressing N^IC-1, N^IC-2, or N^IC-4 compared to the cells transduced with the empty vector control (N^IC-1: 2.9-fold decrease; N^IC-2: 3.0-fold decrease; N^IC-4: 4.3-fold decrease; P<0.01). The results are combined data from three experiments performed in triplicate.