Loss of Bcl-x_L accelerates the death of Εμ-myc pro-B and pre-B cells in culture but has no impact on their cell cycling. (A) Accelerated apoptosis of Eμ-myc/bcl-x^−/− pro-B and pre-B cells in culture. In vitro cell survival assays were performed with FACS-purified donor-derived pro-B cells (Ly5.2⁺B220⁺c-Kit⁺sIg⁻) or pre-B cells (Ly5.2⁺B220⁺c-Kit⁻sIg⁻) from the bone marrow of pre-leukemic mice 8-12 weeks after hematopoietic reconstitution (generated as shown in Figure 2A). The cells were cultured in the absence of cytokines (simple medium) for the indicated periods and cell viability measured by staining with PI plus annexin V-FITC, followed by FACS analysis. Viable cells were defined as PI⁻/annexin V⁻. Data represent mean ± SEM from at least 3 independent experiments with cells of each genotype derived from 3-5 reconstituted mice. Because of the rapid death of Eμ-myc/bcl-x^−/− pro-B cells in culture, times after 24 hours were not examined. *Significantly faster death (P < .05) of Eμ-myc/bcl-x^−/− than Eμ-myc B lymphoid cells. (B) Bcl-x_L loss does not affect the increased cycling of pre-leukemic Εμ-myc B lymphoid cells Donor fetal liver–derived pro-B cells (Ly5.2⁺B220⁺c-Kit⁺sIg⁻) and pre-B cells (Ly5.2⁺B220⁺c-Kit⁻sIg⁻) were FACS sorted from the bone marrow of pre-leukemic reconstituted mice (generated as shown in Figure 2A). Sorted cells were fixed in 70% ethanol and stained with propidium iodide to quantify DNA content. The indicated proportions of cells in the G₀/G₁ and S/G₂/M stages of the cycle were determined by FACS analysis and quantification using manual gating. Data shown are representative of 3 independent experiments.