Figure 4.

Loss of Anp32b impairs in vivo progression of CML and maintenance of LSCs. (A) Total number of WBCs in PB and Wright-Giemsa staining of PB smears were conducted 15 days after transplant from recipients received BCR-ABL1–transduced Anp32b^+/+ and Anp32b^−/− Lin⁻ BM cells (n = 5). (B-C) Representative FACS profiles and percentages of GFP⁺ cells and GFP⁺Gr-1⁺ cells in PB (B) and BM (C) 15 days after first transplantation (n = 5). (D) Survival curves for recipients transplanted with BCR-ABL1–transduced Anp32b^+/+ and Anp32b^−/− Lin⁻ BM cells (n = 6). (E) Survival curves for recipients receiving Anp32b^+/+ and Anp32b^−/− GFP⁺ leukemia cells (1 × 10⁶) on secondary transplantation (n = 4). (F-G) Gross pathology (i) and relative weights (ii) (F) and hematoxylin-eosin staining (G) of the spleens, lungs, and livers from recipients received BCR-ABL1–transduced Anp32b^+/+ and Anp32b^−/− Lin⁻ BM cells at 15 days after transplant (n = 5). (H-I) FACS analysis of GFP⁺LSK cells in BM (H) or spleen (I) at 15 days after first transplantation. Representative FACS profiles (i) and the percentage of GFP⁺LSK cells (ii) are shown (n = 5). (J) Colony-forming assays were performed with BM GFP⁺LSK cells sorted from recipients receiving BCR-ABL1–transduced Anp32b^+/+ and Anp32b^−/− Lin⁻ BM cells at 12 days after transplant. Colony numbers were calculated at day 6 after plating (n = 3). (K) First plated leukemia cells were further collected and used for second plating at day 6. The numbers of colonies were calculated 7 days after plating (n = 3). (L) Cell cycle analysis of GFP⁺LSK cells in BM at 15 days after first transplantation Representative FACS profiles (i) and percentages of cell cycle distributions (ii) are shown (n = 4). (M) Cell division tracing of BM GFP⁺LSK cells. GFP⁺LSK cells were sorted from recipients received BCR-ABL1–transduced Anp32b^+/+ and Anp32b^−/− Lin⁻ BM cells at 15 days after transplant and stained with CellTrace Violet dye. After culturing for 24 hours, cell divisions were analyzed by flow cytometry, and the number of generations is shown. Percentages of cells in each generation were calculated (n = 3). G, generation. (N) Apoptosis analysis of GFP⁺LSK cells in BM 15 days after first transplantation. Representative FACS profiles (i) and the percentage of GFP⁺LSK cells (ii) are shown (n = 5). Error bars denote mean ± SEM. Statistical significance was determined by a 2-tailed, unpaired Student t test (A-C, F, H-N) or log-rank test (D-E). All animal experiments were repeated at least twice with similar results, and the results of 1 representative experiment are shown.