Suppressive effects by Plzf depletion on clonogenicity of Tg LT-HSCs immortalized by conditional expression of MLL-ENL. (A) Structure of the retroviral vector, pMXsU6-KO, carrying the expression cassette of shRNA (short thick line) driven by the mouse U6 (mU6) promoter and KO driven by the PGK promoter in the self-inactivated backbone. Δ, deletion of a fragment containing enhancer elements in the U3 sequence; Ψ, packaging signal. (B) Experimental strategy using immortalized cells with depletion of Plzf by retroviral transduction of shRNA and KO coexpressor. (C) Evaluation of Plzf depletion by shRNA (shP01 or shP21) using the MLL-ENL–immortalized WT KSL (ME) cells. ME cells were retrovirally transduced with Plzf (ME+Plzf) or mock (ME+IP) followed by drug selection. The shRNA-transduced ME+Plzf cells at the first replating were assessed on intracellular FACS. Cells transduced with the shRNA expressor confirmed by KO expression (black line) are shown in the upper panels, where gray shadows represent parental ME+Plzf cells. Plzf expression in shRNA-transduced ME+Plzf and ME+IP cells (as references) is depicted with red thick lines and blue shadow profiles, respectively, in the lower panels, where the gray shadows represent staining with the isotype control antibody in shRNA-transduced ME+Plzf cells. (D-E) Expression levels of Plzf by QRT-PCR (D) and relative colony-forming units (CFU) (E) of the cells sorted from the shRNA-transduced immortalized Tg LT-HSCs in colony replating assays. (F-H) Expression levels of Hoxa9, Meis1, and Evi1 by QRT-PCR (F) and quantification of Gr-1^low/– (G) and apoptotic (H) subpopulations by FACS in the Plzf-depleted immortalized LT-HSCs at the first replating. shLuc, shRNA against luciferase; shP01 and shP21, different shRNAs against Plzf. The bar graphs show the mean ± SD of 3 independent experiments.