Article Figures & Data
Figures
- Fig. 1.
Expression of a Notch1-IC–ER fusion protein in BL41-P3HR1 and HH514 cells. Extracts from hygromycin-resistant single-cell clones derived after transfection of HH514 (A) and BL41-P3HR1 (B) cells with a plasmid encoding an mNotch1-IC–ER fusion protein were separated by gel electrophoresis, Western blotted, and analyzed by immunostaining with a monoclonal antibody specific for an epitope within the hormone binding domain of the ER. Extracts were prepared from HH514-NoER cl3 to cl7 cultivated in the presence of estrogen and from BL41-P3HR1-NoER cl31 and cl33 grown in both the absence and presence of estrogen. An extract from the untransfected BL41-P3HR1 cell line was included as a control. The arrow head marks the position of the band specific for the mNotch1-IC–ER fusion protein.
- Fig. 2.
Activation of a promoter reporter construct containing a multimerized RBP-Jκ binding site by an mNotch1-IC–ER fusion protein in the presence of estrogen. Cells from the parental cell lines HH514 and BL41-P3HR1 and from two HH514-NoER and two BL41-P3HR1-NoER clones, which have been shown to express the mNotch1-IC–ER fusion protein (Fig. 1), were transiently transfected with a promoter reporter construct containing a multimerized RBP-Jκ site. As a positive control, a single-cell clone derived from BL41-P3HR1 expressing an ER-EBNA2 fusion protein (BL41-P3HR1-5E) was included in the experiment. After transfection, each cell suspension was divided in two parts and cultivated with and without estrogen for 2 days. Subsequently luciferase activities (RLU) were determined. The bars represent arithmetic mean values of the promoter activation by estrogen (RLU with estrogen/RLU without estrogen) from at least three (two for parental cell lines) independent experiments. Positive standard deviations are indicated by lines.
- Fig. 3.
The stably expressed mNotch1-IC–ER fusion protein induces the LMP1 promoter in an estrogen-dependent manner. (A) Cells from two HH514-NoER clones, two BL41-P3HR1-NoER clones, and the parental cell lines were transiently transfected with an LMP1 promoter-luciferase construct. As a positive control, a single-cell clone derived from BL41-P3HR1 cells expressing an ER-EBNA2 fusion protein (BL41-P3HR1-5E) was included in the experiment. After transfection, cells were cultivated in the presence or absence of estrogen. Luciferase activities were determined as RLU 2 days after transfection. The bars represent arithmetic mean values of the promoter activation by estrogen (RLU with estrogen/RLU without estrogen) from at least three (two for parental cell lines) independent experiments. Positive standard deviations are indicated by lines. (B) The endogenous LMP1 mRNA expression was analyzed by Northern blotting. The cell lines indicated were cultivated in parallel with and without estrogen. RNAs were separated, blotted, and hybridized with a probe specific for LMP1. (C) The LMP1 mRNA expression of one single HH514-NoER clone (clone 6) was analyzed by Northern blotting in the presence and absence of estrogen and cycloheximide (CHX) as indicated. (B and C) The intensities of the LMP1 signals were quantified and standardized (the weakest measurable signal was set to 1) and are listed below the blots. As a control for RNA quantity and integrity, the ethidium bromide-stained RNA gels are shown.
- Fig. 4.
The stably expressed mNotch1-IC–ER fusion protein induces LMP2A transcription in an estrogen-dependent manner. (A) Cells from two HH514-NoER clones, two BL41-P3HR1-NoER clones, and the parental cell lines were transiently transfected with an LMP2A promoter-luciferase construct. A single-cell clone derived from BL41-P3HR1 expressing an ER-EBNA2 fusion protein (BL41-P3HR1-5E) was included as a positive control. After transfection, each cell suspension was divided in two parts and cultivated with and without estrogen for 2 days. Subsequently luciferase activities (RLU) were determined. The bars represent arithmetic mean values of the promoter activation by estrogen (RLU with estrogen/RLU without estrogen) from at least three (two for controls) independent experiments. Positive standard deviations are indicated by lines. (B) Endogenous LMP2 mRNA expression was analyzed by Northern blotting. ER/EB2-5 cells were cultivated in the presence (+) or absence (−) of estrogen for 4 days. HH514-NoER cells were routinely kept without estrogen. The mNotch1-IC–ER fusion protein was induced by addition of estrogen for 15 or 48 h. Where indicated, cells were grown in the presence of cycloheximide (CHX) for 16 h. RNAs were prepared, separated on a 1% agarose gel, blotted, and hybridized with a probe specific for LMP2. Bands specific for LMP2A and LMP2B are marked by arrowheads. The band marked by the square might represent an antisense transcript. The intensities of the LMP2A signals were quantified and standardized (the weakest measurable signal was set to 1) and are listed below the blot. As a control for RNA quantity and integrity, the ethidium bromide-stained RNA gel is shown at the bottom.
- Fig. 5.
Notch1-IC upregulates CD21 but not CD23 in stably transfected BL cells. (A) CD21 and CD23 expression was analyzed in the cell lines indicated by flow cytometry before (dotted line) and after (thick line) treatment with estrogen for 48 h. (B) Endogenous CD21 mRNA expression was analyzed by Northern blotting. RNAs were prepared from the cells indicated which had been cultivated in the absence (−) or presence (+) (48 h) of estrogen. RNAs were separated, blotted, and hybridized with a probe specific for CD21. The intensities of the CD21 signals were quantified and standardized (the weakest measurable signal was set to 1) and are listed below the blot. As a control for RNA quantity and integrity, the ethidium bromide-stained RNA gel is shown at the bottom.
- Fig. 6.
In Burkitt's lymphoma cells, both Igμ and c-myc are transcriptionally downregulated by the estrogen-activated mNotch1-IC–ER fusion protein. (A) sIgM expression was analyzed in the cell lines indicated by flow cytometry before (dotted line) and after (thick line) treatment with estrogen for 48 h. Endogenous Igμ (B) and c-myc (C) mRNA expression was analyzed by Northern blotting. Cell lines were cultivated without (−) or with (+) estrogen for 48 h. RNAs were separated, blotted, and hybridized with probes specific for the Igμ or c-myc. The intensities of the Igμ and c-mycsignals were quantified and standardized (the weakest measurable signal was set to 1) and are listed below the blots. As controls for RNA quantity and integrity, the ethidium bromide-stained RNA gels are shown at the bottom. In the c-myc blot, RNA quantity of the BL41-P3HR1-5E control (− and + estrogen) was reduced ∼2-fold compared to the other cell lines.
