Deletion of Anp32b reduces HSC numbers by promoting quiescence. (A) Relative Anp32b mRNA levels were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in mouse BM LT-HSCs (Lin⁻Sca-1⁺c-Kit⁺Flk2⁻CD34⁻), ST-HSCs (Lin⁻Sca-1⁺c-Kit⁺Flk2⁻CD34⁺), MPPs (Lin⁻Sca-1⁺c-Kit⁺Flk2⁺), CLPs (Lin⁻Sca-1^lowc-Kit^lowCD16/32⁺Flk2⁺), CMPs (Lin⁻Sca-1⁻c-Kit⁺CD16/32⁻CD34⁺), MEPs (Lin⁻Sca-1⁻c-Kit⁺CD16/32⁻CD34⁻), GMPs (Lin⁻Sca-1⁻c-Kit⁺CD16/32⁺CD34⁺), myeloid cells (Mac-1⁺/Gr-1⁺), B cells (B220⁺), and erythroid cells (TER119⁺) (n = 3). (B) Anp32b deletion was evaluated by genotyping in peripheral blood mononuclear cells (PBMCs) from Scl-Cre⁻;Anp32b^fl/fl and Scl-Cre⁺;Anp32b^fl/fl mice at indicated times after tamoxifen treatment. (C) ANP32B deletion was evaluated by Western blot in total BM cells and Lin⁺ and Lin⁻ cells from 8- to 10-week-old Anp32b^+/+ and Anp32b^−/− mice. (D-F) Representative fluorescence-activated cell sorter (FACS) profiles (D), frequencies (E), and absolute cell numbers (F) of LSK cells (Lin⁻Sca-1⁺c-Kit⁺), LT-HSCs, ST-HSCs, and MPPs in BM from 8- to 10-week-old Anp32b^+/+ and Anp32b^−/− mice (n = 7). (G-I) Frequencies of LK (Lin⁻Sca-1⁻c-Kit⁺), CMPs, GMPs, MEPs (G), CLPs (H), and erythroid cells, myeloid cells, T cells (CD3E⁺), and B cells (I) in BM from 8- to 10-week-old Anp32b^+/+ and Anp32b^−/− mice (n = 7). (J) Total number of BM cells was calculated in 8- to 10-week-old Anp32b^+/+ and Anp32b^−/− mice (n = 11). (K-L) Cell-cycle analysis of LSK cells (K) and LT-HSCs (L) in BM from 8- to 10-week-old Anp32b^+/+ and Anp32b^−/− mice (n = 4). Representative FACS profiles (i) and percentages of cell cycle distributions (ii) are shown. (M) Cell division tracing of LT-HSCs sorted from 8- to 10-week-old Anp32b^+/+ and Anp32b^−/− mice. LT-HSCs were stained with CFSE and cultured for 4 days. Cell divisions were multicolored, and the number of cell divisions is shown. Percentages of cells in each generation were calculated (n = 3). G, generation. (N) Proliferation assay of LSK cells and LT-HSCs in Anp32b^+/+ and Anp32b^−/− mice. Mice were intraperitoneally injected with BrdU and fed with BrdU-containing drinking water for 2 days. Representative FACS plots (i) and the statistical analysis of BrdU-positive cells in LSK cells and LT-HSCs (ii) (n = 5). Error bars denote mean ± SEM. Statistical significance was determined by a 2-tailed, unpaired Student t test (E-N). All animal experiments were repeated at least twice with similar results, and the results of 1 representative experiment are shown.