Etv6^R355X/+ HSPCs show reduced long-term serial repopulation capacity. (A) Schematic diagram describing the competitive BM transplantation assay used for this study. Percent of CD45.2⁺ peripheral blood cells (B), B220⁺CD19⁺ B cells (C), Gr1⁺ myeloid cells (D), CD4⁺ T cells (E), and CD8⁺ T cells (F) after primary transplantation of 10 000 CD45.2⁺Etv6^+/+ (green) or Etv6^R355X/+ (red) LSK cells along with 10 000 CD45.1⁺Etv6^+/+ LSK cells into lethally-irradiated recipient mice (n = 10-12 recipients per genotype per experiment). Data are representative of 3 experiments and show mean ± SD. (G) Percent of CD45.2⁺ peripheral blood Gr1⁺ myeloid cells, B220⁺ B cells, CD4⁺ T cells, and CD8⁺ T cells at 6 months after competitive transplantation. Percent of total BM cells (H), splenocytes (I), peripheral blood cells (J), and LSK cells (K) that are CD45.2⁺ at 6 months after transplantation within remaining recipients (note: half of the recipients were used for serial transplantation depicted in panel N). Data are representative of 3 experiments and show mean ± SD. (L) Schematic diagram describing the LSK cell homing assay used for this study. (M) Frequency (left) and absolute number (right) of CD45.2⁺ LSK cells in the BM of recipients at 18 hours after adoptive transfer. Data are representative of 2 independent experiments and show mean ± SD. (N) Schematic description of the serial BM transplantation assay used for this study. (O) Percent of CD45.2⁺ peripheral blood cells after the secondary transplantation of 200 000 CD45.2⁺Etv6^+/+ (green) or Etv6^R355X/+ (red) BM cells, harvested from primary recipient mice, along with 200 000 fresh CD45.1⁺Etv6^+/+ BM cells, into lethally-irradiated recipient mice (n = 9 recipients per genotype). Data are representative of 2 experiments and show mean ± SD. (P) Percent of CD45.2⁺ peripheral blood cells 4 weeks after tertiary transplantation of 200 000 CD45.2⁺Etv6^+/+ (green) or Etv6^R355X/+ (red) BM cells harvested from secondary recipients, along with 200 000 fresh CD45.1⁺Etv6^+/+ BM cells, into lethally-irradiated recipient mice. Data are representative of 2 experiments (total n = 10 recipients per group) and show mean ± SD. ∗P < .05; ∗∗P < .001; and ∗∗∗P < .0001, determined by multiple unpaired t tests.