FIG 3.

Zfp521^−/− mice possess reduced frequencies of early B cell progenitors. (A and B) qRT-PCR of Zfp521 (A) and Ebf1 (B) in total B220⁺ BM cells and pre-pro-B (Hardy fraction A [FrA]), pro-B (FrB), early pre-B (FrC), late pre-B (FrD), immature (FrE), and recirculating mature B (FrF) cells in wild-type mice. Transcript amounts were normalized to internal Hprt levels and then to levels in total B220⁺ cells. Statistics were generated using one-way ANOVA. Each biological replicate includes three technical replicates. Mouse n: B220⁺, 11 (A) or 10 (B); FrA and FrB, 6; FrC and FrD, 5 (A) or 4 (B); FrE and Fr F, 4 (A) or 3 (B). (C) Transcript levels of Ebf1 were measured in FACS-purified pre-pro-B (FrA), pro-B (FrB), and early pre-B (FrC) cells by qRT-PCR from 3-week-old Zfp521-deficient mice (n = 2) and control littermates (n = 3 to 5). Transcript levels were normalized to internal Hprt levels. Statistics were generated using two-way ANOVA. Each biological replicate includes three technical replicates. (D and E) Frequencies of B220⁺ cells of singlets (D) and numbers of B220⁺ cells (E) in BM of Zfp521^−/− (KO) and control (Ctrl) mice. Cells were gated on Lin^neg (NK1.1, Ly6C, CD317, CD3e, CD11b, CD11c, Gr1, Ter119) cells. Mouse n = 10 Ctrl, 9 KO. (F to H) Flow cytometric analysis of B cell progenitor populations in the BM of Ctrl and KO mice. Mouse n: 4 Ctrl and 4 KO for FrA to FrC, 13 Ctrl and 8 KO for FrD to FrF. (F) Pre-pro-B (FrA), pro-B (FrB), and early pre-B (FrC) cells, gated on Lin^neg B220⁺ CD43⁺ BM cells. (G) Late pre-B (FrD) cells were gated on total B220⁺ BM cells. (H) Immature (FrE) and recirculating mature B (FrF) cells were gated on total B220⁺ BM cells. (I and J) Percent indicated B cell progenitor population of B220⁺ CD43⁺ cells (I) or total B220⁺ cells (J). (K and L) Numbers of indicated B cell progenitor populations in the BM. *, P < 0.05; **, P < 0.01; *** and ###, P < 0.001;**** and ####, P < 0.0001.