PIM2 is required to counter mitochondrial apoptosis. (A) Analysis of cell death was performed in the model previously described.⁵ Cell viability (number of caspase 3–active negative [alive cells] and positive [death cells]) and cell differentiation (number of P3 cells, P2/CD23⁺ cells, P2/CD23^– cells, and P1 cells) were assessed by using flow cytometry. (A, left) The total numbers of dead cells and living cells in the 4 populations on D6. The pie chart shows the percentage of cells, n = 7. (A, right) On D4, cells were treated (or not) with QVD-OPH. On D6, the numbers of cells in each population were recorded. Data are presented as the mean ± SD, n = 6. (B) CASP3 mRNA expression in the various cell populations on D6. The data are presented as the median (range), n = 6. (C) Factors in the mitochondrial apoptosis pathway involved in B-cell differentiation. The green “+” symbol represents a death-activating effect, and the red “-” symbol represents a death-inhibiting effect. The pro-apoptotic protein BAD causes depolarization of the mitochondria, followed by the cleavage of procaspase 9 into active caspase 9, which in turn cleaves procaspase 3 to generate an intermediate, inactive p20 form. Following an auto-catalytic process, the p20 form gives rise to catalytically active p19 and p17 fragments. These molecules cleave PARP (the end effector in the apoptotic process) and also p20 in a feedback loop. Molecules are released from the mitochondria after depolarization and block the action of the inhibitor of apoptosis proteins, including XIAP. The phosphorylation of BAD leads to its cytoplasmic sequestration by 14-3-3, thus hijacking its pro-apoptotic effect. (D and F) Assessment of the protein expression of factors involved in the mitochondrial apoptosis pathway during primary B-cell differentiation. (E) Immunoprecipitation of PIM2 in PBs. PIM2 and BAD were detected by immunoblotting. (G) Immunoprecipitation (IP) of XIAP (top) and caspase 3 (bottom) in PBs. XIAP and caspase 3 were detected by immunoblotting. (H-I) Flow cytometry evaluation of the percentage of active caspase 3–positive PBs on D6, after treatment with SSO-PIM2 (H) or with increasing doses of PIMi (I), relative to controls. Data are presented as the mean ± SD, n = 8. (J) One representative result of the tetramethylrhodamine methyl ester (TMRM) signal on D6 in PBs (analyzed by flow cytometry) after treatment with SSO-PIM2 (left) or PIMi (right), compared with controls. (K-L) Assessment of protein expression levels in PBs on D6 for factors involved in the mitochondrial apoptosis pathway after PIM2 inhibition by SSO-PIM2 (K) or PIMi (10 µM) (L), compared with controls. Statistical significance was evaluated by using Mann-Whitney U (panels A and B), Wilcoxon (panel H), and Friedman (panel I) tests. *P < .05, **P < .01, ***P < .001, ****P < .0001. Cl, cleaved; DMSO, dimethyl sulfoxide; FSC, forward scatter; NBC, naive B cells; ns, not significant; NT, no treatment. Further details are presented in supplemental Figure 4.