fev is expressed and functions in human primitive HSCs. (A) Flow-sorting and purity detection of CD34⁺ and CD34⁻ fractions from CD34-enriched CB cells. (B) RT-PCR detection of fev expression in fractions of (A). (C) Construction of fev-knockdown vectors (iFev or control virus [Ctr.V]). (D) Efficiency validation of fev-knockdown by Western-blot analysis in 293T cells permanently expressing fev. Mock: lysis of 293T cells. (E) The scheme of in vitro functional assays. CB CD34⁺ cells transduced with Ctr.V or iFev lentivirus were cultured in stem cell expansion medium for 5 d and then GFP⁺ cells were flow-sorted for CFC assay. (F) Flow-sorting and purity detection of GFP⁺ cells from 5-d cultured cells of (C). (G) RT-PCR detection of fev in the flow-sorted cells of (D), indicating the efficiency of fev knockdown. (H) Results of methylcellulose CFC assay of Ctr.V and iFev cells. iFev cells had an 8.5-fold decrease in total colonies (124.5 ± 29.9 vs 16.4 ± 3.4, n = 5 independent experiments, P = 0.0008). (I) The proportion of progenitors in total colonies, including erythroid CFU-E (colony forming unit-erythroid), myelo-monocytic CFU-G (colony forming unit-granulocyte), CFU-M (colony forming unit-macrophage), and CFU-GM (colony forming unit-granulocyte, macrophage), and mixed CFU-GEMM (colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte) (n = 5 independent experiments; erythroid: P = 0.4; myelo-monocytic: P = 0.06; mixed: P = 0.0006). (J) Results of CFC replating of primary colonies of (F). iFev cells showed an 11.8-fold decrease in total colonies (32.7 ± 4.8 vs 2.8 ± 3.2, n = 5 independent experiments, P = 0.003). (K) Long time culture-initiating cell frequency of Ctr.V and iFev cells measured by limit-dilution assay. iFev cells showed a 12.7-fold decrease (60.5 ± 21.8 vs 4.8 ± 5.6, n = 5 independent experiments, P = 0.004). PC, positive control in HEK293T cells stably expressing fev; NC, non-template control.