Fig 2: TCF3 induces expression of NOTCH1 and CDKN1A in cHL cell linesExpression of multiple genes in cHL cells expressing pRTS-GFP or pRTS-E47. Cells were treated with DOX for 2 days. RNA was prepared as described in the Materials and methods section. mRNA expression was measured by qRT-PCR. The data represent mean ± SD of three experiments. * p<0.05.

Fig 3: TCF3/E2A induced expression of BIKBIK is an E47 target gene in cHL cell lines. Expression of BIK in cHL cells expressing pRTS-GFP or pRTS-E47. Cells were treated with DOX for 2 days. RNA and protein were prepared as described in the Materials and methods section. A. BIK mRNA expression was measured by qRT-PCR. * p<0.05. B. BIK protein in E47-expressing cells was detected by immunobloting, in comparison with empty vector-expressing cells. The data are representative of three independent experiments with similar results.

Fig 4: E47 induces cell death in cHL cell linesA. KM-H2 and SUP-HD1 cell lines conditionally expressing E47 were established using the pRTS vector system. After transfection, cells were selected with hygromycin for two weeks (KM-H2) and one week (SUP-HD1), more than 95% cells were GFP positive upon DOX treatment. Expression of E47 protein was measured by immunobloting using anti-TCF3 antibody. Anti-ACTB antibody was used as a loading control. * p<0.05 (expression levels upon DOX treatment compared with that of samples without DOX treatment) as it was assessed by double sided T-test. B. Cells transfected with empty pRTS vector (pRTS-GFP) or pRTS-E47 were seeded at low density (0.1 × 106 cells per well in 3 ml of complete medium). DOX was added on day 0 and day 3. On day 2, day 4 and day 6, the cells were counted by flow cytometer and viable cells were discriminated using side/forward scatter parameters. C. The nuclear fragmentation was measured by Nicoletti method after 4 days of incubation with DOX. * p<0.05 as it was assessed by double sided T-test. D. The influence of E47 on cell cycle progression. Cells were seeded at density of 0.5×106 per 10 ml of complete medium and treated with 0.5 µg/ml DOX. After 2 days, cell cycle distribution was measured by PI staining as described in the Materials and Methods section. Experiments were repeated three times. Results are presented as mean ± SD. * p<0.05.

Fig 5: TCF3 negatively regulates proliferationE47 was over-expressed in L428 and UHO1 cells using the lentiviral SF-LV-cDNA-EGFP vector. A. E47 protein levels were analyzed by immunoblot. The percentage of GFP+ cells determined by flow cytometry was about 40%. Anti-ACTB antibody was used as a loading control. B. Percentage of GFP+ cells expressing E47 or empty vector (SF-LV) was measured at the indicated time points by flow cytometry. Percentage on the third day of infection (day 3) was defined as 100%. The data shown are representative of three independent experiments.