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. 2015 Jun 29;212(7):1109–1123. doi: 10.1084/jem.20132100

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© 2015 Ungerbäck et al.

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Figure 1. — Combined transheterozygous loss of Pax5 and Ebf1 results in a partial block at the pro–B cell stage and lineage plasticity in CD19⁺ cells. (A) Graphs displaying the absolute numbers of 7AAD⁻ pro–B cells (Lin⁻B220⁺CD19⁺CD43^highIgM⁻), pre–B cells (Lin⁻B220⁺CD19⁺CD43^low/negIgM⁻), and IgM⁺ B cells (Lin⁻B220⁺CD19⁺CD43^low/negIgM⁺) in bone marrow from Wt (n = 15), Pax5^+/− (n = 10), Ebf1^+/− (n = 7), and Pax5^+/−Ebf1^+/− (TH) (n = 6) mice. (B) Q-PCR analysis of Ebf1 and Pax5 expression in pro–B cells from WT and Pax5^+/−Ebf1^+/− (TH) mice. Each square indicates a biological replicate analyzed by triplicate Q-PCR reactions. Mean of all mice is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. (C) Graphs illustrating the percent of donor (CD45.2⁺) of CD19⁺Thy1.2⁻, CD19⁻Thy1.2⁺, NK1.1⁺, and Gr1/Mac1⁺ cells in spleen of Rag1^−/− CD45.1⁺ recipient mice 6–9 wk after transplantation with 2 × 10⁶ sorted bone marrow CD19⁺IgM⁻ cells from Wt (n = 8) or Pax5^+/−Ebf1^+/− (TH; n = 9) mice. Mice were from two independent transplantation experiments (4 Wt and 4 TH in experiment one and 4 Wt and 5 TH transplantations in experiment two). (D) Graphs and representative FACS plot illustrating the percent of IgM⁺, IgM⁺IgD⁺, and CD43^highIgM⁻ pro–B cells out of CD45.2⁺CD19⁺ from Rag1^−/− mice as described in A. (E) Diagrams and representative FACS plot illustrating the percent of donor (CD45.2⁺) of CD4⁺CD8⁻, CD4⁻CD8⁺, CD4⁺CD8⁺, and Thy1.2⁺Tcrβ⁺ cells in the spleen of Rag1^−/− mice. (F) Southern blot of generated PCR products analyzing immunoglobulin heavy chain VDJ recombination using genomic DNA from Wt pro–B, mouse ear, or sorted TCRβ⁺ cells from TH transplanted Rag1^−/− mice. (G) Diagrams illustrating total CD45.2⁺ out of live cells in thymus, also the percent of donor (CD45.2⁺) of CD19⁺, NK1.1⁺, CD4⁺CD8⁻, CD4⁻CD8⁺, and CD4⁺CD8⁺ in the thymus of Rag1^−/− mice as described in A. In A, C, E, and G, each open circle/square represents one mouse (Wt, n = 4; TH, n = 5) and the data are presented as median (horizontal line) ± interquartile range. Statistical analysis was performed using Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.