Article Figures & Data
Figures
- Fig. 1.
Tetracycline-regulated mNotch1-IC expression in EREB2-5 cells. (A) Extracts from two hygromycin-resistant single-cell clones derived after transfection of EREB2-5 cells with a tetracycline-regulated, Flag-tagged mNotch1-IC (EREB-Notch cl.1 and cl.4) were Western blotted and analyzed by immunostaining with the anti-Flag monoclonal antibody M2. Extracts were prepared in the presence and absence of estrogen and tetracycline. (B) EREB2-5 cells and cells from EREB-Notch clones 1 and 4 were transiently transfected with a promoter-reporter construct containing a multimerized RBP-Jκ binding site. As negative control, a promoter-reporter gene construct without an RBP-Jκ binding site was used. Cells were grown in the presence of estrogen and presence or absence of tetracycline. Four hours before transfection, cells were washed to withdraw estrogen. After transfection, the cells were cultivated for 2 days in the presence (+E) or absence (−E) of estrogen to regulate the function of EBNA2 and in the presence (+N) or absence (−N) of tetracycline to regulate Notch1-IC expression. Luciferase activities were determined as relative light units (RLUs). Mean values and standard deviations of two independent experiments are indicated.
- Fig. 2.
mNotch1-IC stably expressed in EREB2-5 cells cannot maintain B-cell proliferation. After estrogen withdrawal, the same number of viable EREB and EREB-Notch clone 1 and clone 4 cells in the presence or absence of tetracycline (N), leading to the absence (−N) or presence (+N) of Notch1-IC were seeded in 96-well plates, and the number of viable cells was determined by staining with MTT reagent on the days indicated after estrogen withdrawal. As a positive control, the OD values of the parental line EREB in the presence of estrogen (+E) are shown. At day 5, fresh medium was added to all cells. To compare different MTT assays, OD values at day 1 were normalized to 1. Mean values and standard deviations are shown for triplicates.
- Fig. 3.
mNotch1-IC stably expressed in EREB2-5 cells cannot induce LMP1 expression. Protein extracts were prepared from EREB-Notch clones 1 and 4 in the presence and absence of estrogen and tetracycline. The protein extracts were Western blotted and analyzed by immunostaining with monoclonal anti-LMP1 antibody S12.
- Fig. 4.
Tetracycline-regulated mNotch1-IC expression in 1194 cells. (A) Extracts from two hygromycin-resistant single-cell clones derived after transfection of 1194 cells with a tetracycline-regulated, Flag-tagged mNotch1-IC (1194-Notch clones 1 and 2) were Western blotted and analyzed by immunostaining with the anti-Flag monoclonal antibody M2. Extracts were prepared in the presence and absence of tetracycline. The antibody detected a protein with the expected molecular mass of about 70 kDa, whereas an unspecific signal was visible in all lanes at about 80 kDa. (B) Extracts from the controls EREB-pHEBo and 1194-pHEBo and from the 1194-Notch clone 1 in the presence and absence of estrogen and tetracycline were analyzed by Western blots using monoclonal anti-LMP1 antibody S12.
- Fig. 5.
In the absence of estrogen, 1194-Notch cells continue to proliferate for 4 days. (A) 1194-Notch clone 1 cells were kept in the absence of estrogen for up to 7 days, and the number of viable cells was determined by MTT conversion on the days indicated. The rate of MTT conversion at day 0 was set to 1, and OD values were normalized accordingly. (B) After estrogen withdrawal, the same number of viable 1194-Notch clone 1 cells in the presence or absence of estrogen (E) and tetracycline (N), leading to the presence or absence of EBNA2 (+E and −E, respectively) and Notch1-IC (+N and −N, respectively), were seeded in 10-ml cell culture flasks. Cell growth was determined by counting the number of living cells on days 0, 2, 4, and 7 in triplicates. Dead cells were excluded by trypan blue dye staining. (C) The viability of the cells was determined as the ratio of the number of living to total cells. Average values of four experiments are shown. The standard deviations never exceeded 24% of the respective single values.
- Fig. 6.
1194-Notch cells pass through the cell cycle for 6 days. 1194-Notch (A) and 1194-pHEBo (B) cells were stained with propidium iodide 0, 3, 4, 6, 9, or 13 days after estrogen withdrawal. The relative numbers of cells in the G1 and G2phases of the cell cycle were determined by FACS analysis.
- Fig. 7.
mNotch1-IC cannot upregulate c-Myc expression in 1194 cells. Extracts from 1194-pHEBo and 1194-Notch cells (clone 1) grown in the presence and absence of tetracycline were prepared 0, 1, and 2 days after estrogen withdrawal. The protein extracts were Western blotted and analyzed by immunostaining with the anti-Myc monoclonal antibody 9E10.
