Expression of survivin protein and mRNA in G₀ CD34⁺ cells isolated by Hoechst 33342/pyronin Y staining before and after 12 hours' growth factor stimulation.

(A) Hoechst 33342/pyronin Y staining of UCB CD34⁺cells before and after incubation for 12 hours with 100 ng/mL each Tpo, SCF, and FL. The left panel represents the gate for fresh G₀ CD34⁺ cells (R1). Fresh G₀ cells (R1) were incubated with Tpo, SCF, and FL for 12 hours, restained with Hoechst 33342/pyronin Y, and G₀ cells isolated by FACS sorting (middle panel; gate R2). In replicate cultures, R1 cells were prestained with CFSE before incubation, and cell division in the unseparated cell population was analyzed after 12 hours' culture (right panel). Data represent 1 of 5 identical experiments. (B) Postsort analysis of Hoechst 33342/pyronin Y and CFSE staining on cells from the R1 and R2 gates in Figure 2A. Data represent 1 of 5 identical experiments. The R1 and R2 gates were set so that the 20% dimmest pyronin Y cells were collected for fresh and cultured CD34⁺cells. The gates for the postsort analysis were adjusted for the characteristic progressive loss of pyronin Y fluorescence with time.27 33 (C) Intracellular Ki-67 protein expression and DNA staining of fresh G₀ cells (R1) and G₀ cells isolated after culture for 12 hours with growth factors (R2) from Figure 2A. The percentage of cells negative for Ki-67 expression, that is, below isotype staining (horizontal bar), is shown. Data represent 1 of 3 independent experiments. (D) Intracellular survivin protein expression in fresh G₀ CD34⁺cells (R1) and in G₀ cells (R2) from Figure 2A, harvested after 12 hours' incubation with growth factors. Mean channel fluorescence (MCF) of survivin and the percentage of cells staining positive (above isotype control [horizontal bar]) for survivin are shown below each blot. Data represent 1 of 3 identical experiments. (E) Survivin expression was quantitated in the Ki-67–negative fraction of fresh or cultured G₀ cells from Figure 2C. The MCF for survivin and the percentage of survivin-positive cells (above isotype control [horizontal bar]) are shown beneath the blot. Data represent 1 of 3 identical experiments. (F) Total RNA from G₀ cells before (R1) and after (R2) growth factor incubation was subjected to real-time RT-PCR to quantify survivin and Ki-67 mRNA expression. GAPDH was used as the internal control. Because Ki-67 expression of both samples was extremely low, G₁CD34⁺ cells were used to verify the PCR reaction of Ki-67. The y-axis represents ΔRn, which indicates the magnitude of the signal generated at each cycle. The x-axis shows the reaction cycle. Data represent 1 of 2 identical experiments.