Forced expression of ZNF423 leads to a maturation arrest in the B cell lineage in vivo. (A) Structure of MYs retrovirus driven by long terminal repeat (LTR) sequence, venus fluorescence reporter controlled by internal ribosomal entry site (IRES). Scheme of in vivo experiments. CB CD34⁺, cord blood CD34⁺ stem/progenitor cells; IT, intratibial transplantation. (B) Down-regulation of EBF-1 target genes under forced ZNF423 expression in vivo. Venus positive cells from BM were FACS sorted, and mRNA was quantified by qPCR (2^−ΔCt*1000) for n = 17 control and n = 15 ZNF423 positive mice from n = 3 independent experiments. Values were normalized to B2M. Horizontal lines indicate mean. Error bars represent SD. Significance is calculated by Student’s t test (**, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant). (C) Contour plots of FACS-based immunophenotyping of venus⁺ BM cells from two representative animals with similar engraftment. Percentage represents positive cells in the venus⁺ population (Q2). (D) ZNF423⁺ cells show a significant decrease in surface IgM expression. FACS-based immunophenotyping of venus⁺ cells in the BM of control (n = 18) and ZNF423 (n = 17) mice from n = 3 independent experiments. Values represent the percentage of CD34⁺, CD33⁺, CD19⁺, or IgM⁺ cells in the venus⁺ population. Horizontal lines indicate mean. Error bars represent SD. Significance is calculated by Student’s t test (***, P ≤ 0.001; n.s., not significant).