Nfix is required for the persistence of HSPCs ex vivo. (A) Schematic of lentiviral vector used for delivery of shRNAs. MiR30-embedded shRNAs are driven from an MSCV promoter. A downstream PGK promoter drives expression of the fluorescent reporter, mCherry. (B) 293T cells were transduced with FLAG-tagged–Nfix 48 hours prior to transduction with shRNAs. Both transduction steps were carried out with an multiplicity of infection to acquire a transduction frequency of 100%, confirmed by flow analysis. Seven days posttransduction of shRNAs, cells were harvested and analyzed via western blot analysis for Flag and Actin protein levels. (C) LSK cells purified from WBM were transduced independently with 2 distinct shRNAs targeting Nfix, cultured under serum-free conditions for 3 days, flow sorted for mCherry⁺ and then analyzed via qRT-PCR for relative Nfix transcript. (D-H) LSK cells were transduced with either control- or Nfix-shRNAs and cultured ex vivo in serum-free medium supplemented with hematopoietic cytokines for 12 days. (D) Cell growth, (E) viability, (F) percentage of mCherry⁺ in total cell population, (G) percentage of LSK cells in mCherry⁺ population, and (H) % mCherry⁺ cells in LSK cells was assessed every other day. Data represent mean ± standard deviation (SD) from 3independent experiments. (I) Analysis of apoptosis in cells harvested 10 days posttransduction after staining for lineage markers, Sca-1, c-Kit, Annexin V, and DAPI. Data represent mean ± SD from 3 independent experiments. (J) mCherry⁺ LSK cells were recovered by FACS from LSK cells transduced with control- or Nfix-shRNAs and cultured for 7 days ex vivo. These cells were replated in semi-solid methycellulose-based medium supplemented with hematopoiesis-promoting cytokines and hematopoietic colonies were scored 10 days postplating. Data represent mean ± SD from 3 independent experiments. P values were considered statistically significant when *P < .05, **P < .01, and ***P < .001.