Possible involvement of microRNA in Prf1 and GzmB expression in human NK cells. (A-B) In vitro–differentiated human mNK cells were freshly incubated in the absence (mock) or presence of IL-15 (30 ng/mL) after 24-hour deprivation of IL-15. The cells were harvested at the times (0, 6, 12, 24, and 48 hours) indicated relative to IL-15 addition. (A) Kinetic analysis of Prf1 and GzmB expression in human NK cells during activation. (Top) Immunoblot (IB) analyses. (Middle) DNAs amplified by semi-qPCR were stained with ethidium bromide and photographed using N-Trans illuminator. (Bottom) Real-time qPCR (red bars) and band intensities of Prf1 and GzmB proteins (top) were normalized and plotted as a percentage of the maximum (100%; blue bars). The quantitative data are expressed as the mean value of independent measurements from 3 separate experiments (mean ± SEM). (B) Kinetic analysis of cytotoxicity in human NK cells during activation. (C-D) Suppression of Prf1 and GzmB expression by microRNA. Transfected mNK cells with Ago2 and Dicer1 siRNAs were incubated in the absence (C) or presence (D) of IL-15, respectively, and then subjected to IB analysis, real-time qPCR, and standard chromium (Cr) release assay at effector to target cell ratio 5:1 (ET5) and ET1.5. Data are representative of 3 independent experiments (mean ± SEM of triplicates; Student t test, **P < .01 and ***P < .001 vs Ctrl_siRNA).