Survivin expression in UCB CD34⁺ cells sorted by cell cycle.

(A) Fresh UCB CD34⁺ cells were sorted by Hst and PY staining as described in “Materials and methods” into G₀ (R1) and G₁ (R2) cell populations. Data are representative of 3 UCB samples. (B) Cells sorted in panel A were subjected to RT-PCR (40 cycles) and intracellular staining for survivin. Identical results were observed with the use of either an antisurvivin mAb (shown) or a polyclonal antisurvivin antibody. Upper panel: intracellular staining for survivin in fresh G₀CD34⁺ (R1) and G₁ CD34⁺ (R2) following cell sorting by Hst/PY. The area below the horizontal bar in each dot blot represents isotype control. The MCF of FITC-survivin is shown below each dot blot. Lower panel: 50 ng total RNA was used for reverse transcription and one tenth of the cDNA was used for PCR. Lane C was loaded with RT-PCR sample run without any RNA template. Data are representative of 3 UCB samples. (C) UCB CD34⁺ cells cultured with Tpo, SCF, and FL for 48 hours were stained with Hst and PY and sorted into gates R1 through R4 representing G₀, G₁, S, and G₂/M populations as described in “Materials and methods.” All cells were counterstained with anti-CD34 mAb and gated to include only CD34⁺ cells. Data are from 1 of 3 identical experiments. (D) Cells in each gate in panel C were analyzed by multivariate flow cytometry with PI and FITC-antisurvivin mAb. The area below the horizontal bar in each dot blot represents isotype control. MCF of FITC-survivin is shown below each dot blot. Data are from 1 of 3 experiments with identical results. Identical results were observed with the use of FITC-antisurvivin polyclonal antibody. (E) RT-PCR (40 cycles) for survivin and GAPDH mRNA in sorted cell populations from panel C. Lane C was loaded with RT-PCR sample run without any RNA template. (F) Semiquantitative RT-PCR for survivin and GAPDH mRNA in UCB CD34⁺ cells in G₀ and G₁ before and after growth-factor stimulation. Gates were set exactly as in Figures 4A and 4C. Lane 1: freshly isolated G₀ CD34⁺ cells. Lane 2: freshly isolated G₁ CD34⁺ cells. Lane 3: G₀ CD34⁺ cells after 48 hours' stimulation with Tpo, SCF, and FL. Lane 4: G₁ CD34⁺ cells after 48 hours' stimulation with Tpo, SCF, and FL. Lane C was loaded with RT-PCR sample without any RNA template.