Survivin expression in normal hematopoietic cell populations.
(A) Survivin expression in UCB mononuclear cells (MCs), CD34−(lin+) cells (CD34−), and isolated CD34+ cells (CD34+). Cells were stained with PI (x-axis) and FITC–mouse IgG1 (upper panels) or FITC–anti-human survivin (lower panels) (y-axis). Mean channel fluorescence (MCF) of FITC signals is presented in each multivariate dot plot. Preadsorption of the antisurvivin monoclonal antibody (mAb) with excess human survivin/HIS for 30 minutes prior to addition to fixed and permeabilized cells reduced survivin detected by greater than 92% (panel A, right insert), confirming the specificity of the intracellular staining. Cell-cycle status of each cell population analyzed by ModFit software is shown below the corresponding plots. RT-PCR results (35 cycles) for survivin and GAPDH mRNAs from the same cell samples are presented. Lane 1: MC-UCB mononuclear cells. Lane 2: CD34−(lin+) cells. Lane 3: CD34+ cells. Lane 4: C, control sample without any RNA. Data are from a single UCB sample and representative of 5 experiments. (B) Survivin expression in adult peripheral blood mononuclear cells (PBMCs), adult bone marrow mononuclear cells (BMMCs), CD34−(lin+) cells (CD34−), and isolated CD34+. Peripheral blood and bone marrow are from the same donor. The upper panels represent isotype controls, and the lower panels represent survivin protein. Cell-cycle status of each cell population analyzed by Modifit software is shown below the corresponding multivariate dot plot. RT-PCR results (35 cycles) for survivin and GAPDH mRNAs from the same cell samples are presented. Lane 1: PBMCs; Lane 2: BMMCs; Lane 3: CD34−(lin+) cells; Lane 4: CD34+ cells; Lane 5: C, control sample without any RNA. Data are from a single donor and representative of 2 identical experiments with different donors. (C) Survivin mRNA in adult bone marrow CD34+ cells, CD34− cells, and PBMCs from 5 healthy donors. RT-PCR (35 cycles) for survivin and GAPDH mRNAs are shown. C represents control sample without RNA.
