Neither Notch1-IC nor Notch2-IC can maintain B-cell immortalization in the absence of EBNA2. Cells were deprived of estrogen, and Notch1-IC, Notch2-IC, CAT, and EBNA2 were immediately induced by addition of doxycyline and estrogen, respectively. (A) The maintenance of immortalization in Notch1-IC/EREB (Notch1-IC + Dox), Notch2-IC/EREB (Notch2-IC + Dox), and CAT/EREB (CAT + Dox; CAT-Dox) cells in the absence of estrogen was investigated by counting living cells over a period of 8 days after withdrawal of estrogen. CAT/EREB cells after readdition of estrogen (CAT-Dox + estrogen) were used as positive control. Fresh culture medium was added every other day. CAT/EREB cells in the presence of estrogen were diluted when the cells reached a density of 10⁶ cells/mL. These dilutions were included in the calculation of the depicted cell numbers. (B) BrdU incorporation was used as a marker for proliferation. Three days after estrogen withdrawal, the indicated cell lines Notch1-IC/EREB (N1), Notch2-IC/EREB (N2), and CAT/EREB (CAT) were incubated with BrdU for 4 hours. BrdU incorporation into ΔNGFR⁺ cells was analyzed by FACS. Apoptotic cells were determined 3 days after estrogen withdrawal by annexin V/7-AAD staining. Percentage of early apoptosis was measured by analyzing annexin V⁺7-AAD⁻ cells in ΔNGFR⁺ cells of Notch1-IC/EREB (N1), Notch2-IC/EREB (N2), and CAT/EREB (CAT). As positive control, CAT/EREB cells, which were reinduced by estrogen immediately after estrogen withdrawal, were analyzed (EBNA2). Both diagrams show mean values of 4 independent experiments. P values were determined by Student t test comparing Notch1-IC/EREB (N1), Notch2-IC/EREB (N2) in the absence of estrogen and CAT/EREB in the presence of estrogen (EBNA2) with the control CAT/EREB (CAT) in the absence of estrogen.