Figure 4. Effects of Alkbh5 depletion on self-renewal/repopulation of leukemia stem/initiating cells (LSCs/LICs) and normal hematopoiesis.

(A-C) BM lin- cells from Alkbh5 WT or homozygous KO mice were transduced with MA9-YFP retrovirus and then transplanted into primary recipient mice. Five mice from each transplant group were euthanized at the same time (day 42 post-BMT) for LSC/LIC analysis. (A) Representative flow cytometry analysis of LSCs/LICs in BM. (B) Percentage of leukemic GMPs (LGMPs) in YFP⁺ cells. (C) Percentage of apoptotic LGMPs.

(D and E) YFP⁺ BM cells were sorted out from primary BMT mice and transplanted into secondary recipient mice for 2^nd BMT assay. (D) Survival curves of the 2^nd BMT mice (MA9-WT, n=12; MA9-KO, n=10). (E) WBC count from the 2^nd BMT mice euthanized on day 25 post-BMT.

(F) The in vivo limiting dilution assay (LDA). Secondary recipients (n=5 for each group) were transplanted with different doses of BM cells collected from primary recipients (see Figure 2F) euthanized at the same time (Day 61 post BMT).

(G-N) Effects of Alkbh5 KO on mouse static normal hematopoiesis. (G) Regularly bred 6- to 10-weeks Alkbh5 WT (n=7) and KO (n=10) mice were included for the analysis. PB CBC counts were shown in (H). BM analysis of 4 pairs of the same sex/age littermates were shown in (I-N).

(O-R) The in vivo competition assay. (O) Schematic outline of experiment strategy of the in vivo competition assays. (P) Ratio of CD45.2⁺/CD45.1⁺CD45.2⁺ in the PB of recipients. (Q and R) Ratio of CD45.2⁺/CD45.1⁺CD45.2⁺ in the Lin⁺, Lin⁻, LK, LSK, HSC and other progenitor cell populations (Q) and differentiated cell compartments (R) in the BM of recipients.

*p < 0.05; **p <0.01; ***p <0.001; t test (for Figures 4B–C, E, H–N, P–R; Mean±SD values are shown) or log-rank test (for Figures 4D and F). See also Figure S4.