Moz^+/− B-cell progenitors display reduced proliferative capacity. (A) Enumeration of colony formation in methylcellulose culture after plating of unfractionated BM cells and FACS-purified pro–B-cells. (B) Growth curves of pre–B-cells cultured on OP9 cells in the presence of IL-7. After collection by FACS, 500 pre–B-cells were plated and passaged every 5 days. (C-E) Assessment of the survival of (C) pro-B, (D) pre-B, and (E) surface IgM/IgD-positive (sIg+) B cells. At each time point, the proportions of viable cells were determined by staining with propidium iodide and Annexin-V. (F) β-galactosidase activity in spleens from Moz^+/− and control Moz^+/+ mice (n = 6 to 8 per genotype). Scale bars = 100 μm. (G) Quantification of β-galactosidase activity in pre–B-cells of the indicated genotypes cultured over 25 days. At passages 2, 3, and 4, cells were stained for B-cell markers (B220 and CD19), and incubated with a fluorescent substrate for the β-galactosidase enzyme (C₁₂FDG). β-galactosidase activity in pre–B-cells was quantified using flow cytometry. (H-J) Levels of Ink4a, Arf, Ink4b, and p21 mRNA levels in pre–B-cells, (H) ex vivo, (I) at passage 2, and (J) at passage 4. Insufficient Moz^+/− and WT pre–B-cells were available at passage 4 for analysis. Data are presented as mean ± SEM. Asterisks shown in (A,H-J) indicate a statistically significant difference between Moz^+/− and WT mice, or between Moz^+/−; Eμ-Myc^T/+ and Moz^+/+; Eμ-Myc^T/+ mice at *P < .05, **P < .01, and ***P < .001. Gene expression levels of Ink4a, Arf, Ink4b, and p21 in (H-J) were standardized to housekeeping genes Hsp90ab1 and Gapdh, and expression in WT samples was designated 1.