Figure 2

Nap1l3 downregulation impairs maintenance of murine HSCs and blood lineage regeneration. (a) qPCR analysis of Nap1l3 mRNA levels (normalised to Hprt) of sorted LSK HSCs transduced with an shRNA against Nap1l3 (Nap1l3 shRNA), or a control vector (SC shRNA). The data is represented as the mean ± s.e.m, *p < 0.05, ***p < 0.005 (unpaired t-test), n = 3. (b) Flow cytometric analysis of the percentage of CD45.1⁺ donor cells resulting from transduction of sorted cKit⁺ HSPCs transduced with Nap1l3 shRNA (Nap1l3 shRNA) or a control vector (SC shRNA) in peripheral blood of lethally radiated recipient mice, at two, five, eight and 16 weeks post transplantation. The data is represented as the mean ± s.e.m., **p < 0.01, ***p < 0.005 (unpaired t-test), n = 3. (c) Representative flow cytometric chart showing percentage of LSK HSC BM CD45⁺ donor cells, transduced with a negative control vector (SC shRNA, upper panel), or an shRNA vector targeting Nap1l3 (Nap1l3 shRNA, lower panel) in recipient mice five weeks post transplantation. The percentage of LSK HSCs is highlighted in each flow cytometry chart. (d,e) Percentage of donor derived LSK HSCs transduced with shRNAs against Nap1l3 or a control vector. The cells were isolated from the bone marrow of recipient mice 5 weeks (c) and 16 weeks (d) after transplantation and analysed by flow cytometry. **p < 0.01 (unpaired t-test), n = 3. (f–i) Percentage of mature donor cells; CD11b⁺ myeloid (f), Gr-1⁺ granulocytes (g), NK1.1⁺ NK cells (h) and CD19⁺ B cells (i) after transduction of sorted cKit⁺ HSPCs transduced with shRNAs against Nap1l3 (Nap1l3 shRNA) or a control vector (SC shRNA). Bone marrow cells were isolated and analysed by flow cytometry 16 weeks post transplantation. ns = non-significant, *p < 0.05, (unpaired t-test).