Figure 1.
Loss of functional EBF1 results in reduced levels of Myc transcripts and cell cycle arrest in pro-B cells. (A) Schematic drawing of the basic experimental layout. Neonatal Ebf1−/− BM or Ebf1−/− FL were transduced with EBF1-MIG (EBF1) or an EBF1-estrogen receptor fusion protein (EBF1-ER) construct. Cells were then exposed to 4-OHT to allow nuclear translocation of EBF1 in Ebf1-deficient cells carrying the EBF1-ER construct, which drives development into the CD19+ stage. 4-OHT was then withdrawn to test the dependency of EBF1 in the generated CD19+ cells. (B) Number of live cells of cultured EBF1 or EBF1-ER transduced and 4-OHT–treated Ebf1−/− BM cell cultures (as in panel A). Cells were either incubated continuously with 4-OHT (+4-OHT) or with 4-OHT withdrawn (-4-OHT) for 48 to 72 hours as indicated. Mean and standard deviation (SD) are shown; n = 4, from 4 culture experiments. (C) Cell cycle distributions of the cells in panel B at 48 hours after 4-OHT withdrawal. Mean and standard error of the mean are shown, n = 6). (D) The experimental protocol used to test the cell autonomous role of EBF1 in pro–B cell survival in vivo. Ebf1−/− FL cells were transduced to express EBF1 or EBF1-ER and treated with 4-OHT for 14 days. At day 15, a total of 1 million green fluorescent protein (GFP)+CD19+ cells were transplanted into C57BSJL (CD45.1) sublethally irradiated recipients. Donor reconstitution (CD45.2+GFP+), as well as CD19 expression, was determined by FACS 3 weeks posttransplantation. (E-F) Relative cell counts, from the BM of mice transplanted with either EBF1- or EBF1-ER–transduced cells. Mean and SD are shown; EBF1 n = 8, EBF1-ER n = 11, from 2 independent experiments. (G) Diagrams with quantitative reverse transcription PCR data from in vitro expanded Ebf1−/− FL pro-B control cells (FL) or BM cells from EBF1-deficient mice driven to B-cell progenitor stages with conventional or ER-fused EBF1 protein cultured in the presence or absence of 4-OHT for 72 hours. Mean and SD are shown; n = 4 to 7, from 2 independent experiments. For panels C and E-G, each dot indicates a data point, and the statistical analysis is based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Loss of functional EBF1 results in reduced levels of Myc transcripts and cell cycle arrest in pro-B cells. (A) Schematic drawing of the basic experimental layout. Neonatal Ebf1−/− BM or Ebf1−/− FL were transduced with EBF1-MIG (EBF1) or an EBF1-estrogen receptor fusion protein (EBF1-ER) construct. Cells were then exposed to 4-OHT to allow nuclear translocation of EBF1 in Ebf1-deficient cells carrying the EBF1-ER construct, which drives development into the CD19+ stage. 4-OHT was then withdrawn to test the dependency of EBF1 in the generated CD19+ cells. (B) Number of live cells of cultured EBF1 or EBF1-ER transduced and 4-OHT–treated Ebf1−/− BM cell cultures (as in panel A). Cells were either incubated continuously with 4-OHT (+4-OHT) or with 4-OHT withdrawn (-4-OHT) for 48 to 72 hours as indicated. Mean and standard deviation (SD) are shown; n = 4, from 4 culture experiments. (C) Cell cycle distributions of the cells in panel B at 48 hours after 4-OHT withdrawal. Mean and standard error of the mean are shown, n = 6). (D) The experimental protocol used to test the cell autonomous role of EBF1 in pro–B cell survival in vivo. Ebf1−/− FL cells were transduced to express EBF1 or EBF1-ER and treated with 4-OHT for 14 days. At day 15, a total of 1 million green fluorescent protein (GFP)+CD19+ cells were transplanted into C57BSJL (CD45.1) sublethally irradiated recipients. Donor reconstitution (CD45.2+GFP+), as well as CD19 expression, was determined by FACS 3 weeks posttransplantation. (E-F) Relative cell counts, from the BM of mice transplanted with either EBF1- or EBF1-ER–transduced cells. Mean and SD are shown; EBF1 n = 8, EBF1-ER n = 11, from 2 independent experiments. (G) Diagrams with quantitative reverse transcription PCR data from in vitro expanded Ebf1−/− FL pro-B control cells (FL) or BM cells from EBF1-deficient mice driven to B-cell progenitor stages with conventional or ER-fused EBF1 protein cultured in the presence or absence of 4-OHT for 72 hours. Mean and SD are shown; n = 4 to 7, from 2 independent experiments. For panels C and E-G, each dot indicates a data point, and the statistical analysis is based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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