PUM1 and PUM2 contribute to murine HSPC expansion. (A) RT-qPCR analysis of mPum1 and mPum2 transcripts in mouse stem/progenitor subpopulations: LT-HSC (long-term HSC, Lin⁻Sca⁺cKit⁺CD34⁻CD150⁺), ST-HSC (short-term HSC, Lin⁻Sca⁺cKit⁺CD34⁺CD150⁺), MPP (Lin⁻Sca⁺cKit⁺CD34⁺CD150⁻), CMP (Lin⁻Sca⁻cKit⁺CD34⁺CD16⁻), MEP (Lin⁻Sca⁻cKit⁺CD34⁻CD16⁻), and GMP (Lin⁻Sca⁻cKit⁺CD34⁺CD16⁺). Results are normalized to Gapdh expression and expressed relative to Pum expression in LT-HSCs. (B-F) LSK cells were transduced with lentiviral vectors encoding the indicated shRNA targeting mPum1 (shmPum1) or mPum2 (shmPum2) or both (shmPum1a+2a) or luciferase as control (shCtrl) (sequences detailed in supplemental Table 8). shRNA/GFP⁺ LSK cells were sorted 2 days after transduction and maintained in liquid culture. (B) Immunoblot analysis of the indicated proteins at day 7 posttransduction. p85-PI3kinase was used as loading control. (C) Cell expansion analysis along time of culture posttransduction (n = 3). (D) Cell cycle analysis upon propidium iodide labeling at day 5 posttransduction (n = 4). (E) Apoptosis analysis at day 7 posttransduction through AnnexinV-PE/7AAD labeling (n = 4). (F) At day 7 posttransduction, shRNA/GFP⁺ output LSK-derived cells were seeded in methylcellulose medium and numbering of CFC was assessed after 7 days (n = 5); representative micrographs of colonies are presented on the right side (×20). (G-I) Murine LSKCD150⁺ cells were transduced with lentiviral vectors encoding shRNA targeting mPum1 (shmPum1-a) or mPum2 (shmPum2-a) or luciferase as control (shCtrl). shRNA/GFP⁺ cells were sorted 2 days after transduction and maintained in liquid culture. (G) Immunoblot analysis of the indicated proteins at day 7 posttransduction. β-Tubulin was used as loading control. (H) Cell expansion analysis along time of culture posttransduction (n = 3). (I) Three days posttransduction, lethally irradiated C57BL/6-Ly5.2 mice received 15 000 sorted shRNA/GFP⁺ LSKCD150⁺ cells from C57Bl/6 (Ly5.1) donors, in competition with the same amount of GFP⁻ LSKCD150⁺ cells from Ly5.1 mice, together with 1.5 × 10⁵ Ly5.2 BM cells. Presence of GFP⁺ cells in CD45.1⁺ BM cells of engrafted mice was assessed 4 months later by flow cytometry (at least 10⁵ events). Each symbol represents data from a single chimeric mouse. (Figure representative of 1 experiment out of 2). Data are expressed as mean ± SEM. *P < .05; **P < .01; ***P < .001 (Student t test).