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Blood, Vol. 113, Issue 22, 5506-5515, May 28, 2009
Notch1, Notch2, and Epstein-Barr virus–encoded nuclear antigen 2 signaling differentially affects proliferation and survival of Epstein-Barr virus–infected B cells
Blood Kohlhof et al.
113: 5506
Supplemental materials for: Kohlhof et al
Files in this Data Supplement:
- Table S1. Primer sequences used for quantitative RT-PCRs (PDF, 36.9 KB)
- Figure S1. Kinetics of doxycycline and estrogen induction (JPG, 229 KB)
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(A) Cells were deprived of estrogen for three days prior to doxycycline addition. Cells were stained with an anti NGF-R antibody 0h, 1h, 2h, 3h, and 4h after doxycyline addition and surface NGFR expression was measured by FACS analysis. The percentage of NGFR+ cells was high immediately after establishment of the stably transfected cell lines (like in this figure), but declined continuously during culture (see Fig. S3). (B) The ER/EBNA2 fusionprotein/RBPJ complex interacts with its binding sequences on the LMP2A promoter 20 minutes after estrogen induction: EREB2–5 cells were cultivated in the absence of estrogen for three days prior to re-addition of estrogen. Nuclear extracts of EREB2–5 cells were prepared at the indicated time points after estrogen addition and incubated with a radioactively labelled oligonucleotide representing the EBNA2 responsive element of the LMP2A promoter encompassing two RBPJ binding sites. Protein/DNA interactions were revealed by an electrophoretic mobility shift assay (EMSA) described in detail in Zimber-Strobl et al. (1994) EMBO J. 13: 4973–4982. The EBNA2 containing complex could be supershifted (see asterisk) by addition of an EBNA2 specific antibody.
- Figure S2. Notch1-IC and Notch2-IC regulate mRNA and protein levels of known target genes similar to EBNA2 (JPG, 208 KB)
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(A) The effect of Notch1-IC, Notch2-IC, and EBNA2 in EREB2–5 cells on the transcriptional regulation of CR2 and IGHM was investigated by qRT-PCR. Cells were deprived of estrogen for three days prior to re-addition of doxycycline and estrogen for the indicated time points. As expected, CR2 is upregulated and IGHM downregulated by Notch1-IC, Notch2-IC, and EBNA2 expression, indicating that the stably transfected Notch1-IC and Notch2-IC expression vectors are functionally active. Values are standardized to the value at 0h to obtain the fold mRNA inductions at different time points after doxycycline or estrogen addition. Data represent the average of triplicate samples. Standard deviations are shown by error bars. *: p < 0.05 calculated by the two-tailed Student’s t test. Color coding is elucidated beneath the diagram. (B) The influence of Notch1-IC, Notch2-IC, and EBNA2 on the surface expression of CD21 and IgM was analyzed by FACS. Notch1/2-IC and EBNA2 expression was induced immediately after estrogen withdrawal by addition of doxycyline or estrogen. Three days later cells were stained with anti-CD21 and anti-IgM antibodies and analyzed by FACS. The histogram shows an overlay of the surface expression of CD21 and IgM as well as of the cell size (Forward scatter) in the absence (blue) or presence (red) of the indicated transgenes.
- Figure S3. MACS purification of NGFR+ cells (JPG, 193 KB)
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NGFR+ cells were purified by MACS (magnetic cell sorting) prior to the preparation of total RNA, which was used for the Affymetrix chip analysis. NGFR+ cells were analyzed before and after the MACS separation by FACS analysis. Numbers indicate the percentage of NGFR+ cells of the shown experiment, numbers in parenthesis indicate the mean values of the percentages of NGFR+ cells from three independent experiments. The cell lines are indicated above the figure: Notch1-IC (Notch1-IC/EREB), Notch2-IC (Notch2-IC/EREB), CAT (CAT/EREB).
- Figure S4. Transcriptome analysis of genes which are stronger induced by Notch1-IC and Notch2-IC than by EBNA2 (JPG, 624 KB)
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DTX1 was chosen as a representative gene for the correlation of genes strongly regulated by Notch1-IC and/or Notch2-IC, but not or only barely by EBNA2. (A) The heat map displays genes showing a high correlation to the expression pattern of DTX1. Absolute expression values are compressed for better comparability to −2 to +2. Red squares indicate a high (up to +2) expression, green (up to −2) a low expression and black the mean (0) expression over the whole kinetic of all cell lines. Gene names as provided by Affymetrix are depicted on the right hand site of the heat map. The cell lines are indicated above the heat map: N1 (Notch1-IC/EREB), N2 (Notch2-IC/EREB), CAT (CAT/EREB), EBNA2 (CAT/EREB after addition of estrogen). (B) All genes displayed in the heat map were ordered by their strength of induction by Notch1-IC 24h after doxycyline addition. (C) Genes from (B) were allocated to functional groups by dCHIP software using GeneOntology annotation (http://biosun1.harvard.edu/complab/dchip). Only functional groups with values of at least 4 (P < 0,001) are shown.
- Figure S5. Transcriptome analysis of genes which are strongly induced by EBNA2 but not by Notch-IC (JPG, 1.01 MB)
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PIK3R1 was chosen as a representative gene for the correlation of genes strongly regulated by EBNA2, but not or only barely by Notch1-IC and/or Notch2-IC. (A) The heat map displays genes showing a high correlation to the expression pattern of PIK3R1. Absolute expression values are compressed for better demonstration to −2 to +2. Red squares indicate a high (up to +2) expression, green (up to −2) a low expression and black the mean (0) expression over the whole kinetic of all cell lines. Gene names as provided by Affymetrix are depicted on the right hand site of the heat map. The cell lines are indicated above the heat map: N1 (Notch1-IC/EREB), N2 (Notch2-IC/EREB), CAT (CAT/EREB), EBNA2 (CAT/EREB after addition of estrogen). We found an enormous number of target genes, which were induced 4 hours after EBNA2 activation and are not regulated by Notch-IC. (B) Genes, which are displayed in the heat map, were ordered by their strength of induction 4 hours after EBNA2 activation. Alignment of the genes according to the magnitude of regulation by EBNA2 pointed out that the strongest difference between EBNA2 and Notch-IC is found in the regulation of chemokine ligands. Thus four chemokine ligands (XCL1, CCL4, XCL2, and CCL3) were in the group of the first ten genes, which were most strongly regulated by EBNA2 and only moderately by Notch-IC. (C) Genes from (B) were allocated to functional groups by dCHIP software using GeneOntology annotation (http://biosun1.harvard.edu/complab/dchip). Only functional groups with values of at least 4 (p < 0,001) are shown. The other numbers indicate the following p values: 5: p < 0,0001; 6: p < 0,00001; 7: p < 0,000001.
- Figure S6. Transcriptome analysis of genes which are similarly induced by EBNA2 and Notch-IC (JPG, 730 KB)
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CR2 was chosen as a representative gene for the correlation of genes induced similarly by EBNA2 and Notch1-IC/Notch2-IC. (A) The heat map displays genes showing a high correlation to the expression pattern of CR2. Absolute expression values are compressed for better demonstration to −2 to +2. Red squares indicate a high (up to +2) expression, green (up to −2) a low expression and black the mean (0) expression over the whole kinetic of all cell lines. Gene names as provided by Affymetrix are depicted on the right hand site of the heat map. The cell lines are indicated above the heat map: N1 (Notch1-IC/EREB), N2 (Notch2-IC/EREB), CAT (CAT/EREB), EBNA2 (CAT/EREB after addition of estrogen). (B) Tabular depiction of the genes from (A).
- Figure S7. Induction of cell cycle and proliferation associated genes by Notch1-IC, Notch2-IC, and EBNA2 (JPG, 1.33 MB)
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The heat map displays genes associated with cell cycle regulation and proliferation, which were at least two fold upregulated by Notch1-IC, Notch2-IC, or EBNA2. Absolute expression values are compressed for better demonstration to −2 to +2. Red squares indicating a high (up to +2) expression, green (up to −2) a low expression and black the mean (0) expression over the whole kinetic of all cell lines. Numbers and bars on the left hand site describe the clusters after hierarchical clustering. Gene symbols and gene names are depicted on the right hand site of the heat map. The cell lines are indicated above the heat map: N1-IC (Notch1-IC/EREB), N2-IC (Notch2-IC/EREB), CAT (CAT/EREB), EBNA2 (CAT/EREB after addition of estrogen). The transcripts whose expression was validated by quantitative RT-PCR are indicated by asterisks.
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