Nfix Expression Critically Modulates Early B Lymphopoiesis and Myelopoiesis
Fig 5
Efficient differentiation of myeloid cells in Nfix expressing cells.
(A) MigR1 and Nfix transduced total BM cells were plated in methylcellulose media (M3434) and colonies scored after 9–11 days according to morphological criteria. The mean percentage of erythroid (E), granulocyte/erythrocyte/monocyte/megakaryocyte (GEMM), granulocyte/monocyte (GM) macrophage (M) and granulocyte (G) colonies are shown from 3 independent experiments +/- SEM. A statistically significant decrease in CFU-EE colonies indicated. (B) Representative flow cytometric analysis of total cells from colony assay in (A). (C) Graph of methylcellulose colony numbers from HSPCs, CMPs and GMPs sorted and transduced with control MigR1 or Nfix. Colonies were counted on day 12 and graph represents mean of triplicate plates normalized to GFP expression +/- SD. (D) Representative flow cytometric analysis of cells from colony assay in (C). 32D cells transduced with MigR1 or Nfix maintained in either IL3 (E) or G-CSF (F) for 5 days, and analyzed for F4/80 expression by flow cytometry. The mean percentages of 3 independent experiments are shown +/- SD. (G) 32D cells and (H) Ba/F3 cells transduced with MigR1 and Nfix and qRT-PCR performed. Graphical presentation of relative mRNA expression of Nfix, MMP9, G-CSFr and C/EBPalpha genes and presented relative to control MigR1 cells. Error bars denote +/- SD of 3 technical replicates. Data representative of 2 biological replicates.