Ectopic expression of MYC rescues pro–B cell expansion in the absence of EBF1. Cell recovery from cultures of FL Ebf1^−/− cells in vitro cultivated as described in Figure 1A. Cells were transduced with an empty MIG-GFP (MIGR1) vector (A), an EBF1-GFP vector (EBF1) (B), or an ER-fused EBF1 protein (EBF1-ER) (C) and serially transduced with an RFP control or a MYC-expressing RFP retrovirus. The diagrams display cell recovery when the cells were grown in the presence or absence of 4-OHT for 72 hours. Mean and standard deviation are shown; n = 6 to 12, from 3 independent experiments. (D) Recovery of CD45.2⁺CD19⁺GFP⁺RFP⁺ BM cells 3 to 4 weeks after transplantation of Ebf1^−/− FL pro-B cells transduced with EBF1-ER and either a control RFP or MYC-expressing RFP-encoding retrovirus into sublethally irradiated CD45.1 mice. Mean and standard deviation are shown; n = 4 to 6 transplanted mice. (E) Representative FACS plot of immunoglobulin M (IgM) and CD19 expression on recipient (CD45.1) and donor (CD45.2⁺GFP⁺RFP⁺) cells. (F) Fraction of early (Annexin V–positive/4′6-diamidino-2-phenylindole–negative [red bar]) and late (Annexin V–positive/4′6-diamidino-2-phenylindole–positive [blue bar]) apoptotic cells 48 hours after removal of 4-OHT in EBF1- and EBF1-ER–expressing Ebf1^−/− BM cells transduced with either RFP-control or RFP-Myc virus. Mean and standard error of the mean are shown; n = 3. The statistical analysis is based on comparisons of data from cells grown in the presence or absence of 4-OHT. (G) Histograms displaying overlays of an FACS staining of MYC protein in different stages of the cell cycle. (H) Bars depicting the ratios of MYC median fluorescent intensity (MFI) of each stage of the cell cycle compared with G₀ 48 hours after 4-OHT withdrawal in Wt and Ebf1^−/−EBF1-ER BM cells Mean and standard error of the mean are shown, n = 6. The statistical analysis is based on a Student unpaired t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, not significant.