Loss of MNT increases apoptosis of pro-B cells in IL-7 cultures. (A) Expression of MYC, MNT, and MCL-1 protein in CD19⁺IgM⁻ cells sorted by FACS from bone marrow of 6-week-old WT mice, before and during culture in IL-7, and 24 hours after IL-7 removal. (B-D) MNT-deficient pro-B cells exhibit increased apoptosis, elevated BIM, and decreased MCL-1. Pro-B cells were obtained by culturing CD19⁺ bone marrow cells (isolated using microbeads) in IL-7 (5 ng/ml) for 4 days. (B) Annexin-V staining of Rag1Cre and Mnt^fl/flRag1Cre pro-B cells; profiles are typical of those from ≥3 mice of each genotype (see panel E). (C) Flow cytometric analysis of intracellular BIM using Bim^−/− cells (gray shaded) as a negative control. Bar graphs compare percentage BIM-positive cells and mean fluorescence intensity (MFI) from WT (light green) vs Mnt^fl/flRag1Cre (light blue) mice, determined in 3 independent experiments; MFI of BIM-null cells was subtracted from that of either WT or Mntf^l/flRag1Cre cells stained in same experiment. (D) Typical western blot of pro-B cells from WT, Mnt^fl/flRag1Cre and control Bim^−/− mice. For each blot, BIM and MCL-1 levels were quantified relative to ACTIN and normalized to WT value. Bar graphs show mean fold-change ± SD **P ≤ .01; ***P ≤ .001. (E) Bim heterozygosity significantly reduces apoptosis of MNT-null pro-B cells. Cells were stained with annexin-V after 4 days in IL-7. Bar graphs show mean ± SD; *P ≤ 0.05; **P ≤ .01; ****P ≤ .0001.