Role of survivin in leukemic NK cell survival. (A) Quantitative real-time RT-PCR was performed to measure levels of survivin mRNA in PBMCs from NK-LGL leukemia patients (CD3⁻CD56⁺ > 80%) or purified NK cells isolated from normal donors. Each circle represents an individual purified NK sample. cDNA samples were diluted 1:100 for 18S expression. ***P < .0005 indicates leukemic NK cells versus normal NK cells (Mann-Whitney test). (B) Suspension splenocytes were isolated from spleens of NK-LGL leukemic F344 rats or age- and gender-matched normal rats. RNAs were harvested from these cells, and quantitative real-time RT-PCR was performed to measure levels of survivin mRNA. cDNA samples were diluted 1:100 for 18S expression. **P < .005 indicates leukemic NK cells versus normal NK cells (Mann-Whitney test). (C) Mitochondria were isolated from human aggressive NK-LGL leukemia cell line NKL or rat aggressive NK-LGL leukemia cell line RNK-16, or pooled enriched NK cells (CD3⁻CD56⁺ 80%-95%) from 5 normal human donors, or 4 individual patients with chronic NK-LGL leukemia (CD3⁻CD56⁺ cells > 80%), then resolved in the sodium dodecyl sulfate polyacrylamide gel electrophoresis gel loading buffer in a boiling water bath for 5 minutes. Western blot analysis was performed for detection of survivin. Cox IV, a mitochondria marker, was used as loading control. (D) NKL cells were transfected with 10 μg control siRNA or human survivin ON-TARGET plus SMARTpool siRNA (Dharmacon) per 5 × 10⁵ cells, each complexed within cationic nanoliposomes, then MTT assay was performed 72 hours after transfection. **P < .005 indicate significant difference in cell viability of survivin siRNA-transfected cells compared with control siRNA-transfected cells (Student t test). Inset, Western blot analysis was performed for survivin in the control siRNA or survivin siRNA-transfected NKL cells 72 hours after transfection. The equal loading of protein was confirmed by probing with glyceraldehyde 3-phosphate dehydrogenase.