Relationship between enforced positioning within IL-7⁺ niches, RAG2 protein stability, and preB cell developmental progression. (A) Overexpression of FAK (Ptk2) or EV reported by GFP in retrovirally transduced BM cells isolated from C57BL/6 mice. FAK expression in preB cells transduced with EV (left) or FAK (middle). Representative histogram of preB cells transduced with FAK compared with proB cells transduced with EV (right). (B) pSTAT5a in EV- or FAK-transduced preB cells. (C) Frequency of EV- (left) or FAK-transduced (right) GFP^Hi BM B-lineage cells and granulocytes from femur and tibia. (D) Overexpression of CXCR4 R334X (WHIM mutation) in retrovirally transduced BM cells isolated from RAG2:GFP knock-in mice. Transduced cell subsets reported by human CD4. CXCR4 expression in EV- (left) or CXCR4^R334X-transduced (right) preB cells gated on hCD4^Lo and hCD4^Hi cells. (E) BrdU incorporation in EV- (left) or CXCR4^R334X-transduced preB cells. Numbers indicate the frequency of BrdU⁺ cells. (F) RAG2:GFP expression in gated hCD4^Hi and hCD4^Lo preB cells (EV, left; CXCR4^R334X, right). (G) Quantification of RAG2:GFP fusion protein geometric mean fluorescence intensity (GMFI). Circles depict individual mice. (H) Frequency of hCD4^Hi B-lineage cells and granulocytes in femur and tibia BM. Circles indicate mean ± SD (n = 3). (C and H) Error bars indicate SD of three independent experiments. Numbers inside histograms (A, B, and D) indicate geometric mean fluorescence intensity ± SD (n = 3 mice in each group). Data in all panels were pooled from three independent experiments. *, P < 0.05; **, P < 0.01 by unpaired Student’s t test (C and H). *, P < 0.05 by paired Student’s t test (G).