Effects of forced expression of wild-type and dominant-negative Survivin constructs on the proliferation of p21^+/+ and p21^–/– CFU-GM and c-kit⁺, Lin^– cells. Marrow cells from p21^+/+ and p21^–/– mice were infected with MIEG3-vector (–), MIEG3-Survivin (W), or MIEG3-T34A Survivin (T34A) in the presence of TPO, FL, and SCF for 48 hours. Following infection, either GFP⁺ cells or GFP⁺, c-kit⁺, Lin^– cells were isolated using FACS. (A) Five thousand GFP⁺ cells were plated in agar in the presence of 10 ng/mL rmGM-CSF and 50 ng/mL rmSCF, and total CFU-GM was quantitated after 7 days. The percentage increase of CFU-GM in p21^+/+ and p21^–/– cells expressing various Survivin constructs was compared with p21^+/+ cells transduced with empty vector. Data are expressed as mean ± SEM of 5 experiments. (B) Fifty thousand GFP⁺ cells were lysed and subjected to Western blot analysis for Survivin expression. The ratio of Survivin to β-actin protein measured by densitometry is shown beneath the blot. (C) Gating criteria for isolation of c-kit⁺, Lin^– cells after retrovirus transduction. GFP⁺, c-kit⁺, and Lineage-marker–(Ter119, B220, GR-1, Mac-1, and CD3) depleted cells (R2 gate in right plot) were collected immediately after infection using FACS. Horizontal and vertical bars in the right plot represent isotype staining. (D) After infection, 10 thousand sorted, transduced GFP⁺, c-kit⁺, and Lin^– cells were cultured in the presence of 10 ng/mL rmGM-CSF and 50 ng/mL rmSCF. Viable cells in each culture were enumerated at 72 and 120 hours by trypan blue exclusion. The percentage increase in viable cell number after growth factor incubation of GFP⁺, c-kit⁺, Lin^– p21^+/+, and p21^–/– cells transduced with wild-type (W) and T34A Survivin were compared with p21^+/+ cells transduced with empty vector. Data represent mean ± SEM of 3 experiments. Absolute viable cell counts in 1 representative experiment are shown in the inset.