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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 2. — Combined heterozygous loss of Pax5 and Ebf1 results in Notch-dependent lineage plasticity in pro–B cells at the single cell level. (A) Representative FACS plots of sorted Wt and Pax5^+/−Ebf1^+/− pro–B cells after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. (B) Cellular composition of clones from 10 Wt, Pax5^+/−, Ebf1^+/−, or Pax5^+/Ebf1^+/− (TH) or single Wt or Pax5^+/−Ebf1^+/− pro–B cells (Lin⁻B220⁺CD19⁺CD43^highIgM⁻) incubated for 14 d on OP9-DL1 cells to stimulate T cell development, with or without the γ-secretase inhibitor DAPT. Total number of wells analyzed from co-culture of OP9-DL1 and 10 pro–B cells are Wt (51), Pax5^+/− (14), Ebf1^+/− (38), and Pax5^+/−Ebf1^+/− (86) and co-culture of OP9-DL1 and one pro–B cell are Wt (12) and Pax5^+/−Ebf1^+/− (15) collected from 2–3 independent experiments. Similarly, the number of wells analyzed after co-culture of OP9-DL1 and 10 pro–B cells in the presence of DAPT are Wt (21), Pax5^+/− (2), Ebf1^+/− (14), Pax5^+/−Ebf1^+/− (17). CD19 cells were scored as CD19⁺Thy1⁻CD3⁻, Thy1 cells as CD19⁻Thy1⁺CD3⁻, and CD3 as CD19⁻Thy1⁺CD3⁺. (C) The graphs display gene expression analyzed by Q-PCR in cultures derived from Wt (4 wells) and Pax5^+/−Ebf1^+/− (4 wells) after 14 d of T cell–inducing co-culture on OP9-DL1 stroma cells. Each square represents one well, analyzed in triplicate Q-PCR reactions. Mean of all wells is presented as a horizontal line. Statistical analysis was performed using unpaired Student’s t test. N.D = no samples in this group showed detectable expression after 45 cycles of PCR. (D) Representative FACS plot of Pax5^+/−Ebf1^+/−(TH) CD43^highIgM⁻ cells cultivated for 31 d on OP9-DL1 supplied with IL-7, kit ligand, and Flt3 ligand day 0–21. On days 22–31, IL-7 was substituted with IL-2 to further stimulate T cell development. The data are representative of three cultures from two experiments. (E) Dot plots representing the cellular content of cell cultures after transduction with either a pMIG (GFP) control or Ebf1 encoding retrovirus (Ebf1-pMIG) in Pax5^+/Ebf1^+/− (TH) pro–B cells after 14 d of co-culture with OP9-DL1. The data are collected from pro–B sorted from five different animals. Statistical analysis was performed using unpaired Student’s t test. **, P < 0.01; ****, P < 0.0001.