Figure 3. Functional analysis of HSCs in KI mice. (A) Flow cytometric enumeration of LSK, GMP, CMP, and MEP cells in the BM of 4-mo-old Vav-Cre–negative Asxl1-MTfl/fl (control) and Vav-Cre–positive Asxl1-MTfl/fl (KI) mice. (B and C) Proportion of LSK, LT-HSCs, short-term (ST) repopulating HSCs, MPPs, Lin−c-Kit+, CMPs, GMPs, and MEPs in both mice (n = 5 in B and n = 8 in C). The data are expressed as relative values (percentage), with whole BM cells being 100%. (D) Flow cytometry analysis of CD41lowCD71high (ErP) and CD41highCD71low (MkP) cells from MEP cell populations (Lin−c-Kit+Sca1−CD34−FcγR−) of primary Vav-Cre–negative Asxl1-MTfl/fl (control, 70 wk old, white circle, n = 5) and Vav-Cre–positive Asxl1-MTfl/fl mice (KI, 70 wk old, black circle, n = 5). The frequency in MEPs is shown. (E) Absolute number of colonies (BFU-E, CFU-E, and CFU-GM) from whole BM cells of Vav-Cre–negative Asxl1-MTfl/wt (control) and Vav-Cre–positive Asxl1-MTfl/wt (KI) mice. For the CFU-E and BFU-E assay, 105 and 2 × 104 BM cells per well were used, respectively. For the CFU-GM assay, 2 × 104 BM cells per well were used. Duplicate samples were plated. (F) Absolute number of colonies (BFU-E and CFU-GM) from 250 LT-HSC and MPP cells per well of Vav-Cre–negative Asxl1-MTfl/fl (control) and Vav-Cre–positive Asxl1-MTfl/fl (KI) mice. Duplicate samples were plated. (G) Absolute number of colonies in the M3434 condition. A total of 2 × 104 whole BM cells per well and 250 LT-HSC or MPP cells per well were used. (H and I) Absolute number of colonies with or without ASXL1 expression in the M3434 condition (H) and the BFU-E, CFU-E, and CFU-GM condition (I). We sorted the Lin− fraction using MACS and retrovirally transduced empty vector (pMYs-IRES-hNGFR) and ASXL1-WT (pMYs-ASXL1-IRES-hNGFR) into Lin− control or KI cells. Colony-formation assay was performed using FACS-sorted hNGFR+ cells. A total of 2 × 104 whole BM cells per well for BFU-E, CFU-GM, and M3434 colonies and 105 cells per well for CFU-E were used. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).
Figure 3.

Functional analysis of HSCs in KI mice. (A) Flow cytometric enumeration of LSK, GMP, CMP, and MEP cells in the BM of 4-mo-old Vav-Cre–negative Asxl1-MTfl/fl (control) and Vav-Cre–positive Asxl1-MTfl/fl (KI) mice. (B and C) Proportion of LSK, LT-HSCs, short-term (ST) repopulating HSCs, MPPs, Lin−c-Kit+, CMPs, GMPs, and MEPs in both mice (n = 5 in B and n = 8 in C). The data are expressed as relative values (percentage), with whole BM cells being 100%. (D) Flow cytometry analysis of CD41lowCD71high (ErP) and CD41highCD71low (MkP) cells from MEP cell populations (Lin−c-Kit+Sca1−CD34−FcγR−) of primary Vav-Cre–negative Asxl1-MTfl/fl (control, 70 wk old, white circle, n = 5) and Vav-Cre–positive Asxl1-MTfl/fl mice (KI, 70 wk old, black circle, n = 5). The frequency in MEPs is shown. (E) Absolute number of colonies (BFU-E, CFU-E, and CFU-GM) from whole BM cells of Vav-Cre–negative Asxl1-MTfl/wt (control) and Vav-Cre–positive Asxl1-MTfl/wt (KI) mice. For the CFU-E and BFU-E assay, 105 and 2 × 104 BM cells per well were used, respectively. For the CFU-GM assay, 2 × 104 BM cells per well were used. Duplicate samples were plated. (F) Absolute number of colonies (BFU-E and CFU-GM) from 250 LT-HSC and MPP cells per well of Vav-Cre–negative Asxl1-MTfl/fl (control) and Vav-Cre–positive Asxl1-MTfl/fl (KI) mice. Duplicate samples were plated. (G) Absolute number of colonies in the M3434 condition. A total of 2 × 104 whole BM cells per well and 250 LT-HSC or MPP cells per well were used. (H and I) Absolute number of colonies with or without ASXL1 expression in the M3434 condition (H) and the BFU-E, CFU-E, and CFU-GM condition (I). We sorted the Lin− fraction using MACS and retrovirally transduced empty vector (pMYs-IRES-hNGFR) and ASXL1-WT (pMYs-ASXL1-IRES-hNGFR) into Lin− control or KI cells. Colony-formation assay was performed using FACS-sorted hNGFR+ cells. A total of 2 × 104 whole BM cells per well for BFU-E, CFU-GM, and M3434 colonies and 105 cells per well for CFU-E were used. Data are presented as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s t test).

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