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. 2016 Jun 7;6:27379. doi: 10.1038/srep27379

Figure 3. HSCs require hnRNP L to be functional.

Figure 3

(a) Differentiation assays on OP9 and OP9-DL1 stromal cells; representative dot plots of myeloid cells (Gr1+Mac1+), B-cells (B220+CD19+) and T-cells (CD4+CD8+) differentiation of FL LSK cells from control (upper panel) and VavCre+hnRNP Lfl/fl mice (lower panel) (n = 3) (b) Colony assay on methylcellulose; 500 LSK cells from FL or BM from the indicated mice were seeded on methylcellulose and colonies were counted after 10 days of culture (n = 3). (c) Non-competitive transplantation assay: 2 × 105 FL cells of either VavCre+hnRNP Lfl/fl or control (hnRNP Lfl/fl) stage E14.5 embryos (both CD45.2+) were transplanted into irradiated syngenic CD45.1+ mice and the percentage of total CD45.2+ cells in the blood were measured at 4, 8 and 12 weeks post-transplantation. (d) The frequency of CD45.2+ cells within the T-, B-, myeloid or erythroid subpopulations (i.e. within CD3, B220, Gr1, Ter119 positive cells) in the blood of the recipient mice 12 weeks post-transplantation was assessed (n = 6). (e) Competitive transplantation assay: 2 × 105 FL cells from VavCre+hnRNP Lfl/fl or VavCre+hnRNP Lwt/fl were mixed with 2 × 105 normal CD45.1+ BM cells at a ratio of 1:1 for a total of 4 × 105 cells. The mixtures were transplanted into irradiated CD45.1+ recipient mice and the percentages of CD45.2+ cells in the blood were measured at 4, 8 and 12 weeks post-transplantation. (f) The frequency of CD45.2+ cells within the T-, B-, myeloid or erythroid subpopulations (i.e. within CD3, B220, Gr1, Ter119 positive cells) in the blood of the recipient mice 12 weeks post-transplantation was assessed (n = 3). (g) 7.5 × 104 Lin−cKit+ progenitor cells from VavCre+hnRNP Lfl/fl or VavCre+hnRNP Lwt/fl were mixed with 2 × 105 normal carrier CD45.1+ BM cells. The mixtures were transplanted into irradiated CD45.1+ recipient mice and the percentages of CD45.2+ cells in the blood were measured at 4, 8 and 12 weeks post-transplantation. (h) The frequency of CD45.2+ cells within the T-, B-, myeloid or erythroid subpopulations (i.e. within CD3, B220, Gr1, Ter119 positive cells) in the blood of the recipient mice 12 weeks post-transplantation was assessed (n = 3). All error bars are means ± SEM.