Mcl-1 plays an essential role in survival of leukemic NK cells and is degraded by FTY720. (A) Increased expression of Mcl-1 in NK-cell leukemia. Proteins were isolated from PBMCs of 5 individual patients with chronic NK-cell leukemia (CD3⁻CD56⁺ cells > 80%), or pooled enriched NK cells (CD3⁻CD56⁺ 80%-95%) from 10 normal human donors, then resolved in the SDS-PAGE gel loading buffer in a boiling water bath for 5 minutes. Western blot analysis was performed for detection of Mcl-1. GAPDH was used as a loading control. (B) Knockdown of Mcl-1 induces apoptosis in leukemic NK cells. Human NKL cells were infected with the concentrated PLKO.1 Mcl-1 shRNA or scramble shRNA lentiviral stocks for 48 hours, and then apoptosis assay was performed. *P < .05 indicates significant difference in apoptosis of Mcl-1 shRNA-infected cells compared with control shRNA-infected cells (Student t test). (Inset) Western blot analysis was performed for Mcl-1 in the control shRNA or Mcl-1 shRNA-infected NKL cells 48 hours after infection. The equal loading of protein was confirmed by probing with GAPDH Ab. (C) Dose-dependent reduced expression of Mcl-1 after FTY720 treatment. Western blot analysis was performed for Mcl-1 after 24-hour treatment of RNK-16 cells or PBMC cells from an NK-cell leukemia patient, with 2.5, 5, and 10μM FTY720. Data are representative of 3 independent experiments on RNK-16 cells or PBMCs from 3 chronic NK-cell leukemia patients. (D) Time-dependent decreased expression of Mcl-1 after FTY720 treatment. Western blot analysis was performed for Mcl-1 after treatment of RNK-16 cells with 10μM FTY720 for 6 and 24 hours, or PBMC cells from an NK-cell leukemia patient with 5μM FTY720 for 2 and 6 hours. Data are representative of 3 independent experiments on RNK-16 cells or PBMCs from 3 chronic NK-cell leukemia patients.