Nfkbiz is a direct target gene for Reg1 and Reg3 that is potent to promote myelopoiesis. (A) Schematic representation for B-cell differentiation of multipotent progenitor (MPP^hId3-ERT2) cells retrovirally transduced with MSCV-Thy1.1 vector. Cells were then cultured under B-cell–differentiation condition. (B-C) In vitro B-cell differentiation in MPP^hId3-ERT2 cells retrovirally overexpressing Nfkbiz and Ehd3 as well as myeloid-lineage TFs. At day 7, cells were analyzed by flow cytometry. Flow cytometry plots of CD19 and CD11b after gated on Thy1.1⁺ cells (B) and percentages of CD19⁺ and CD11b⁺ Thy1.1⁺ cells (C) were shown (n = 6 each). (D) Numbers of CD19⁺CD11b^– and CD19^–CD11b⁺ cells in tetO-cre-Reg1^fl/flReg3^–/– MPP^hId3-ERT2 cells cultured under B-cell–differentiating condition in the presence or absence of DOX at day 6 (n = 6 each). (E-F) Quantitative RT-PCR (E) and immunoblot analysis (F) of Nfkbiz in tetO-cre-Reg1^fl/flReg3^–/– MPP^hId3-ERT2 cells at the indicated time points after DOX treatment (n = 3 each in E). Cells were cultured under stem-cell condition for the indicated time. (G) DOX-inducible overexpression of FLAG-tagged Reg1 (D141N) or Reg3 (D271N) in tetO-cre-Reg1^fl/flReg3^–/– MPP^hId3-ERT2 cells. (H) RNA immunoprecipitation quantitative PCR (RIP-qPCR) analysis of a set of genes from lysates of tetO-cre-Reg1^fl/flReg3^–/– MPP^hId3-ERT2 cells expressing FLAG-tagged Reg1 or Reg3 as in (G) (n = 4 each). Data are representative of 2 (B-D, G-H) or at least 3 (E-F) independent experiments. Data are presented as mean ± SD (C-E,H). Statistical significance was calculated by 1-way ANOVA with Holm-Sidak multiple comparisons test (C,E,H) or unpaired 2-tailed Student t test (D).