RNA editing of Azin1 sustains the differentiation of HSCs. (A) A schematic of the experimental procedure. Briefly, c-Kit⁺ cells were transduced with a GFP-tagged WT (A), non-edited (TC) or edited Azin1 (G) overexpression lentivirus. The GFP⁺ cells were sorted for CFU assay 48 hours after transduction (SFFV and E2A, promoters; AsiSI and MIul, restriction sites). (B) Colony formation of c-Kit⁺ BM cells after transduction with the indicated lentivirus, The number of colonies in the different groups were counted after 7 days of incubation, including Mix, GM, and E (n = 5 per group, 2 independent experiments; Error bars represent SD). (C) Schematic of the transplantation experimental procedure. Briefly, the c-Kit⁺ cells were sorted from the BM of Azin1^fl/fl;ER-Cre mice (CD45.2⁺) and transduced with the 3 different overexpression lentivirus vectors. The infection efficiency was assessed by flow cytometry 48 hours after transduction, and 5 × 10⁵ infected c-Kit⁺ cells were injected into lethally irradiated (9.0 Gy) recipient mice (CD45.1⁺). One month after transplantation, the recipient mice were treated with tamoxifen (1 mg for 5 days) to induce deletion of endogenous Azin1. Then, the reconstitution of donor cells in the peripheral blood was monitored for another 4 months. (D) The relative percentage of GFP⁺ cells in CD45.2⁺ cells in the peripheral blood of recipient mice after first and secondary transplantation (n = 6-8 per group; 3 independent experiments; Error bars represent SEM from 3 replicates). (E-G) Flow cytometry analysis of BM cells from recipient mice 5 months after first transplantation. Flow plots and histograms display the frequencies of HSPCs and HPCs in lineage^– cells (E), stem cell subpopulations (LT-HSC, ST-HSC, and MPP cells) in LSK⁺ cells (F), and progenitor subpopulations (CMP, GMP, and MEP) in LSK^– cells (G) (n = 5-6 per group; 2 independent experiments; Error bars represent SD). (H) The frequencies of stem and progenitor cells in GFP⁺ cells of recipient mice, 5 months after first transplantation (n = 5-6 per group; 2 independent experiments; Error bars represent SD). (I) Stem and progenitor BM cell counts, 5 months after first transplantation (n = 5-6 per group; 2 independent experiments; Error bars represent SD). *P < .05; **P < .01; ***P < .001.