Fig. 2

Lactoferrin deficiency reduces the proportion of stromal cells in the bone marrow microenvironment and decreases CXCL12 expression to inhibit the early development of B cells. a Cells were isolated from the bone marrow of Lf^−/− mice and WT littermates. Frequencies of bone marrow stromal cells (CD45⁻CD31⁻TER119⁻CD106⁺) were identified by flow cytometry. Representative data from 11 specimens each group are shown. b Expression levels of some factors in bone marrow stromal cells sorting from WT and Lf^−/− mice were determined by RT-qPCR. n = 3. c, d ELISA was performed for CXCL12 and IL7 of the supernatant of bone marrow stromal cells (isolated from WT and Lf^−/− mice). e In vitro differentiation experiment: bone marrow cells from Lf^−/− or WT mice were added in 12-well plates, with CXCL12 recombinant protein (10 ng/ml), or PBS as control, for 9 days. The proportions of pro-B cells generated from the bone marrow cells were then determined by flow cytometric analysis. f Different stages of B cells were isolated from the WT and Lf^−/− mouse bone marrow cells, and the surface expression of CXCR4 in each stage of B cells was determined by flow cytometric analysis. Each group has six mice. g, h In vitro differentiation experiment: bone marrow cells from Lf^−/− mice were added in 12-well plates at 5 × 10⁴ cells per well, with (1) PBS, or (2) LY2510924 (10 ng/ml), or (3) CXCL12 recombinant protein (10 ng/ml), or (4) LY2510924 (10 ng/ml) and CXCL12 recombinant protein (10 ng/ml), for 9 days. Each group has six specimens. The proportions of pro-B cells (g) and the phosphorylation levels of AKT and ERK in total B cells (h) generated from the bone marrow cells were then determined by flow cytometric analysis. i In vitro differentiation experiment: bone marrow cells from Lf^−/− mice were added in 12-well plates at 5 × 10⁴ cells per well, with (1) PBS, or (2) MK-2206 2HCL (10 ng/ml), or (3) U0126-EtOH (10 ng/ml), or (4) MK-2206 2HCL (10 ng/ml) and CXCL12 (10 ng/ml), or (5) U0126-EtOH (10 ng/ml) and CXCL12 (10 ng/ml), for 9 days. The proportions of pro-B cells generated from the bone marrow cells were then determined by flow cytometric analysis. j Bone marrow stromal cells transplantation experiment: bone marrow stromal cells from WT or Lf^−/−mice (CD45.2⁺) were mixed with bone marrow cells from WT (CD45.1⁺) mice at a ratio of 1:3, and then the mixed cells were transplanted into the irradiated lethal Lf^−/− mice (CD45.2⁺). After 6 weeks, the recipient mice were killed to prepare the bone marrow single-cell suspension, and the B cell proportion of each stage of B cell differentiation was analyzed by flow cytometry