Gfi1 is essential in LMPP cells for B cell lineage specification.

<p>(A) LMPPs from WT, KI, KD and KO were sorted and cultured on stromal OP9 cells in the presence of Flt3L (5 ng/mL) and IL-7 (10 ng/mL). After 10 or 12 days of co-culture cells were analysed for B220 and CD19 expression. The cell number for each mouse is indicated in bold. (B) Pre-pro B cells were sorted and cultured on OP9 cells in the presence of Flt3L (5 ng/mL) and IL-7 (10 ng/mL). After 10 or 12 days of co-culture, cells were analyzed for B220 and CD19 expression. (C) PCR analysis showing the deletion of <i>Gfi1</i> flox allele in BM B220⁺CD19⁺ B cells and in splenic B220⁺ B cells. B220⁺CD19⁺ BM cells and B220⁺ splenic cells were sorted and lysed to extract DNA. (D) and (E) WT, Gfi1 KO (KO) and MB1-cre <i>Gfi1</i> flox/flox (MB1/Gfi1fl) mice were analyzed for B cells in the BM (D) and spleen (E). (F) Absolute numbers of B220⁺CD19⁺ B cells in WT, Gfi1 KO (KO), <i>Gfi1</i> flox/flox (Gfi1fl), MB1-cre (MB1/WT) and MB1-cre <i>Gfi1</i> flox/flox (MB1/Gfi1fl) BM. (G) Absolute numbers of B220 B cells in WT, Gfi1 KO (KO), <i>Gfi1</i> flox/flox (Gfi1fl), MB1-cre (MB1/WT) and MB1-cre <i>Gfi1</i> flox/flox (MB1/Gfi1fl) spleens. (H) BM cells from Gfi1-GFP mice were gated for HSC, MPP, LMPP, pre-pro B cell (Pre-pro B) and GFP expression (blue line) was measured in each population. (I) Sorted LMPPs from WT, KI, KD and KO mice were cultured on OP9-DL1 stromal cells in the presence of SCF (10 ng/mL), Flt3L (5 ng/mL) and IL-7 (1 ng/mL). After 15 or 21 days of co-culture, cells were analyzed for CD44, CD25, CD4 and CD8 expression. (J) Sorted LMPPs from WT, KI, KD and KO were cultured on OP9 cells in the presence of myeloid cytokine mix as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160344#pone.0160344.ref037" target="_blank">37</a>]. After 10 days, cells were analysed for Gr1 and Mac1 expression by flow cytometry. FACS plots are representative of at least three independent experiments.