Fig. 1.

K3 loss mobilizes LSCs. (A) BA⁺K3^WT and BA⁺K3^KO mixed chimeras generated by transferring BM cells carrying a conditional, HSPC-inducible, and tetracycline-controlled Bcr-Abl (BA) transgene and a conditional, tamoxifen-inducible, Fermt3-null allele wild type mixed with WT BM cells into lethally irradiated recipient mice. The congenic CD45.1 and CD45.2 surface receptors are used to distinguish transgenic, WT, and host cells. Tamoxifen or oil were administered orally (p.o.). (B) Bcr-Abl, c-Abl, K3, and tubulin protein expression in CD45.2⁺ splenocytes. (C) Ratio between transgenic (CD45.2⁺) and WT (CD45.2⁺CD45.1⁺) leukocytes in the PB of BA⁺K3^WT (n ≥ 8) and BA⁺K3^KO (n ≥ 11) mixed chimeras. Statistics by multiple t test, two-sided. (D and E) Numbers of B220⁺ B cellc (D) and Gr1^high/Mac1⁺ neutrophils (E) per microliter of PB determined 2 wk after leukemia induction (n = 18). Statistics by Mann–Whitney U test (D) and unpaired t test, two-sided (E). (F and G) Percentage of PB neutrophils (F) and ratio (G) between transgenic (CD45.2⁺) and WT (CD45.2⁺CD45.1⁺) PB neutrophils in BA⁺K3^WT (n ≥ 8), BA⁺K3^KO (n ≥ 11), and BA⁻K3^WT (n = 7) chimeras at the indicated time points after tetracycline withdrawal. Statistics by multiple t test, two-sided. (H) Ratio between transgenic (CD45.2⁺) and WT (CD45.2⁺CD45.1⁺) BM neutrophils in BA⁺K3^WT and BA⁺K3^KO mixed chimeras 6 wk after tetracycline withdrawal (n ≥ 7). Statistics by unpaired t test, two-sided. (I) Numbers of LSK, LSK CD150⁻CD48⁺, LSK CD150⁺CD48⁺, and LSK CD150⁺CD48⁻ cells per femur in BA⁺K3^WT and BA⁺K3^KO mixed chimeras 6 wk off tetracycline (n ≥ 7). Statistics by unpaired t test, two-sided. (J) Ratio between transgenic (CD45.2⁺) and WT (CD45.2⁺CD45.1⁺) LSK cells in the BM of BA⁺K3^WT and BA⁺K3^KO mixed chimeras 6 wk after tetracycline withdrawal (n ≥ 7). Statistics by unpaired t test, two-sided. (K) Percentage of BrdU⁺ (bromodeoxyuridine-positive) transgenic (CD45.2⁺) LSK cells in the BM of BA⁺K3^WT and BA⁺K3^KO mixed chimeras 6 wk off tetracycline (n = 5). Statistics by unpaired t test, two-sided. (L) Number of LSK cells per microliter of PB in BA⁺K3^WT and BA⁺K3^KO mixed chimeras 2 wk after tetracycline withdrawal (n ≥ 16). Statistics by unpaired t test, two-sided. (M) Frequency of GM-CFUs in 50 µL PB of BA⁺K3^WT and BA⁺K3^KO mixed chimeras 2 wk off tetracycline after 13 d of culture (n ≥ 5). Statistics by Mann–Whitney U test, two-sided. (N) Percentage of apoptotic Annexin-V⁺ PB LSK cells in BA⁺K3^WT and BA⁺K3^KO mixed chimeras 2 wk after tetracycline withdrawal (n ≥ 7). Statistics by unpaired t test, two-sided. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.