Mifepristone Augments uNK Cell-Cytotoxicity Figure 2. Analysis of uNK-cell cytotoxicity and perforin expression after treatment with different concentrations of mifepristone. The purified uNK cells were incubated with 0, 65, 200, and 1000 nmol/L mifepristone for 24 h. Then, they were subjected to analysis of uNK-cell cytotoxicity and perforin expression. A, a representative flow cytometry profiles of perforin expression was shown. Uterine NK-cell cytotoxicity (B) and perforin (C) expression after treatment with different concentrations of mifepristone were evaluated. Values are expressed as means 6 SEM. The influence of mifepristone was evaluated by 4 independent experiments. P-values referred to One-way analysis of variance, n = 6, * P,0.05 vs. control group. doi:10.1371/journal.pone.0036413.g002 er’s protocol. The percentage of cytolysis was calculated as: [12(experimental group OD2effector cell OD)/target cell OD]6100% [25,26], where OD is the optical density. Western blot Because NK cells usually die within 24 h when cultured in vitro, uNK cells were isolated and cultured in the medium without IL-2 for 12 h as a negative control for MAPK/ERK activation. IL-15, known as a strong stimulator of NK cells, was added and used as a positive control for MAPK/ERK activation [27–29]. Uterine NK cells were lysed using protein lysis buffer (2% sodium dodecyl sulfate [SDS], 20% glycerol, 60 mM Tris-HCl) containing protease inhibitor. The cell lysates were centrifuged at 10,0006g for 10 min to remove cell debris. The supernatants were collected and denatured at 95uC for 10 min in 16 SDS loading buffer. Protein samples were diluted in 66 loading sample buffer (50 mM Tris-HCl, 100 mM dithiothreitol, 2% SDS [w/v], 10% glycerol [v/v] and a trace mount of bromophenol), resolved using 10% SDS-PAGE, and then transferred onto nitrocellulose membranes (Amersham Bioscience, Piscataway, NJ, USA). Membranes were blocked in 5% fat-free milk for 1 h and then incubated overnight at 4uC with primary antibodies against extracellular-signal- regulated kinase (ERK), phosphorylated (p)-ERK, p38 MAPK (p38), p-p38, c-Jun N-terminal kinase (JNK), and p-JNK (Cell Signaling, Danvers, MA, USA). The following day, membranes were washed (63, for 10 min each) in PBS containing 0.1% Tween 20 and then incubated for 1 h with the corresponding secondary antibodies at room temperature. Proteins were detected with an enhanced chemiluminescence reagent (Amersham Biosci- ence). Density of the protein bands was measured using Quantity One software (Bio-Rad, Hercules, CA, USA). Data analysis All results were expressed as means 6 SEM. Before statistical analysis, the data were tested for normal distribution by applying the one-sample Kolmogorov-Smirnov test. Homogeneity of variances was evaluated by Levene’s test. Statistical comparisons were performed by one-way ANOVA followed by a least significant difference test. A P-value,0.05 was considered significant. Statistical analyses were performed using SPSS software, version 16.0 (SPSS, Chicago, IL, USA). PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36413