C/EBPα is down-regulated in APL-initiating cells, and its haploinsufficiency increases APL penetrance. (A) Comparison of C/EBPα RNA expression in APL-initiating cells (LICs) and their phenotypic counterpart (wild-type promyelocytes, wt pro) isolated from a wild-type bone marrow (fractions 3 and 4, Figure 2Biii) by quantitative real-time PCR normalized to GAPDH levels (representative of 3 independent experiments, P < .003, a 3-fold decrease for C/EBPα RNA levels). (B) Western blot analysis of C/EBPα protein (middle panel) in CD34⁺, c-kit⁺, FcγRIII/II⁺, Gr1^int cells isolated from wild-type (wt) and APL-initiating cells (LICs; proteins extracted from 20 000 cells were loaded on each lane). Cells isolated from leukemic mice expressed the fusion protein PML-RARα (top panel). Loading was assessed with an antibody against HSP90 (bottom panel). (C) Kaplan-Meier analysis for leukemia-free survival in wild-type (crossed line), C/EBPα^+/− (crossed line), hCathepsinG PML-RARα (PR, hatched line), and C/EBPα^+/− x hCathepsinG PML-RARα mice (PR C/EBPα^+/−, ▵). The cumulative survival was plotted with respect to time in days. The cumulative probability of death resulting from APL was significantly higher in hCathepsinG PML-RARα C/EBPα^+/− compared with hCathepsinG PML-RARα animals (P = .02).