Figure 2. Cell cycle arrest of Ikaros-deficient c-Kit^lo ‘pro-B’ cells despite Igμ expression.

(a, b) Cell cycle analysis. Following intraperitoneal injection of BrdU for 40 minutes, the bone marrow of 5-8-week-old mice was stained for cell surface proteins and subsequently fixed, permeabilized and DNase I-treated prior to detection of total DNA with 7-AAD and incorporated BrdU with BrdU antibodies. c-Kit^hi (upper panel) and c-Kit^lo (lower panel) CD19⁺CD2^–IgM^–IgD^– cells from experimental Cd79a-Cre Ikzf1^fl/– mice (Δ/–; black bars) and control Cd79a-Cre Ikzf1^fl/+ mice (Δ/+; grey bars) were analyzed by flow cytometry for DNA content and BrdU incorporation. Representative FACS plots (a) are shown together with the average percentage of cells in the different cell cycle phases (b). n indicates the number of mice analyzed. Statistical data (b) are shown with SEM and were analyzed by two-way analysis of variance (ANOVA) with Bonferoni’s post-test; *** (p < 0.001). (c) Flow cytometric analysis. c-Kit^hi and c-Kit^lo CD19⁺CD2^–IgM^–IgD^– cells from the bone marrow of experimental Cd79a-Cre Ikzf1^fl/– mice (black line) and control Cd79a-Cre Ikzf1^fl/+ littermates (grey) were analyzed for cell size (FSC-A) and expression of Igμ, λ5 and VpreB by intracellular staining. Small pre-B cells (c-Kit^lo/–CD19⁺CD2⁺IgM^–IgD^–; dashed line) of control Cd79a-Cre Ikzf1^fl/+ mice are shown for comparison.