Targets of miR-21 and the function in NK-cell lymphoma/leukemia. (A) Apoptosis among NKL cells. ASO-21– and SCO-21–treated cells were exposed to 100 μmol/L etoposide for 4 hours. Top panels show flow cytometric analysis of ASO-21–treated and SCO-21–treated NKL cells: y-axis, cells stained by propidium iodide (PI); x-axis, cells stained by annexin V–FITC. Apoptotic cells are in Regions 1 (R1) and 2 (R2). Apoptotic (R1 + R2) cells of ASO-21–treated cells are showing significantly greater percentages than those cells treated with SCO-21. Bottom panel shows the percentages of apoptotic cells (R1 + R2). □ and ■ depict percent apoptosis (R1 + R2) among NKL cells treated with SCO-21 and ASO-21, respectively. Symbols and bars indicate means and SDs of triplicate samples. (B) Cell-cycle analysis for NKL cells treated with ASO-21 or SCO-21. In the top panel, red lines show the cell-cycle pattern of cells treated with SCO-21, whereas green lines show the cell-cycle pattern of those treated with ASO-21. G₁, S, and G₂/M phases are shown as described. Average (n = 3) percentages among SCO-treated NKL cells: G₁, 29.3%; S, 61.3%; and G₂/M, 9.4%. Average (n = 3) percentages among ASO-treated NKL cells: G₁, 25.3%; S, 66.5%; and G₂/M, 8.2%. (C) Western analysis for direct candidate targets of miR-21 of NKL cell line treated with ASO-21 and SCO-21. Northern blot analysis of miR-21 in NKL cells treated with ASO-21 and SCO-21 are also shown. Fold changes in proteins expression are shown below the gels and normalized to the level in NKL SCO-21 treated cells, which are assigned a value of 1.00. (D) Western analysis for candidate downstream targets of miR-21 of NKL cell line treated with ASO-21 and SCO-21. Fold changes in proteins expression are shown below the gels and normalized to the level in NKL SCO-21–treated cells, which are assigned a value of 1.00. Bottom panel shows an immunoblot analysis of p21, p27, and p53 in NKL cells treated with ASO-21 or SCO-21. (E) Incidence of apoptosis among YT cells. In the left panel, cells treated with ASO-21 or SCO-21 were exposed to 100 μmol/L etoposide for 4 hours. □ and ■ depict percent apoptosis among YT cells treated with SCO-21 and ASO-21, respectively. Symbols and bars indicate means and SDs of triplicate samples. Apoptotic cells of ASO-21–treated cells show significantly greater percentages than those cells treated with SCO-21. In the right panel, Western analysis of miR-21 targets in YT cells treated with ASO-21 or SCO-21. Fold changes in protein expression are shown below the gels and normalized to the level in SCO-21–treated cells, which was assigned a value of 1.00. (F) Incidence of apoptosis among transduced MOTN1 and control cells. In the left panel, MOTN1 cells transduced with miR-21 plus GFP or GFP alone (control) were exposed to 100 μmol/L etoposide for 4 hours. □ and ■ depict percent apoptosis among GFP-transduced (control) and miR-21–transduced cells, respectively. Symbols and bars indicate means and SDs of triplicate samples. Apoptotic cells of miR-21 transduced cells show significantly lower percentages than those of control cells. In the right panel, Western analysis of PTEN, pAKT, total AKT, and Bim expression in MOTN1 cells. Fold changes in protein expression are shown below the gels and are normalized to the level in GFP-transduced control cells, which was assigned a value of 1.00.