Scl-directed shRNAs impair reconstitution in vivo and facilitate the G₀-G₁ transition of LT-HSCs in culture. (A) Schematic representation of shRNA lentiviral delivery in lineage-depleted bone marrow cells. Data shown are representative of 2 independent experiments. (B) Western blot of nuclear extracts from lineage-depleted bone marrow cells after the delivery of 3 different Scl-directed shRNAs or a nontargeting control. SC35 was used as loading control. (C) After gene delivery and puromycin selection, cells (CD45.2⁺) were transplanted into irradiated hosts (CD45.1⁺). The bone marrow was analyzed 8 months later for reconstitution (CD45.2⁺) within the HSC population (KSL CD34⁻). Box plots illustrate pooled data from control cells expressing the empty vector or a nontargeting shRNA (NTCtrl) or from cells expressing Scl-directed shRNAs illustrated in panel B (shScl). (D-E) Cell-cycle analyses of shRNA-infected cells were performed using Hoechst 33342 and Pyronin Y. Stacked columns represent the relative proportion of cells in G₀, G₁, and S/G₂/M phases within the LT-HSCs (D) and progenitor populations (E). Data represent the mean ± SEM of pooled data for controls or shScl-expressing cells as in panel C (*P < .01, **P < .001).