Scratched monolayer cells with 200 μl pipette tip, washed cells 3 times with PBS, and added 2 ml medium without FBS into each well. The values of scratch were measured at 0 h and 24 h after scratching by Image Pro-Plus 6.0 system. Transwell migration assay Transwell chambers (8 μm pore size; Millipore, USA) were also used to measure cell migration. Seeded 2 × 105 cells into each upper chamber with 200 μl fresh medium without FBS, added 500 μl medium

GDC-0973 purchase with 20% FBS into each lower chamber, three duplicate wells were set up for each group. After 12 h, fixated cells with methanol for 5 min, and stained cells by hematoxylin for 30 min. Cleaned upper chamber and inverted the chamber, counted cell numbers on the lower membrane under high power lens (× 400) in five random visual fields. Matrigel invasion assay Transwell chamber (8 μm pore size; PI3K inhibitors ic50 Millipore, USA) covered with 100 μl of 1 mg/ml Matrigel

(BD, USA) was used to measure cell invasive ability. Seeded 1 × 105 cells into each upper chamber with 200 μl fresh medium without FBS, added 500 μl medium with 20% FBS into each lower chamber, three duplicate wells were set up for each group. After 12 h, fixated cells with methanol for 5 min, and stained cells by hematoxylin for 30 min. Cleaned upper chamber and inverted the chamber, counted cell numbers on the lower membrane under high power lens (× 400) in five random visual fields. Xenograft model assay The experimental protocol was approved by Zhengzhou University Ethics Committee for Animal PI3K inhibitor Experimentation. Female BALB/c nu/nu mice (4-5 weeks old, 13-17 g) were purchased from Vital River Laboratory Animal Technology Co., Ltd (Peking, China), and were randomly assigned into four groups with 4 mice per group. About 1 × 107 cells were suspended in 0.2 ml PBS and injected subcutaneously into one mouse. The tumors were monitored every 5 days beginning at day 5 by measuring two perpendicular diameters with a caliper. The mice were sacrificed on the 35th day after injection, tumors were dissected and measured, and tumor volume in mm3 was calculated by the formula: volume = (width)2 × length/2 [10]. Statistical analysis Average values were expressed HSP90 as

mean ± standard deviation (SD). Count data were analyzed by χ2 test. Measurement data were analyzed by one-way ANOVA and Bonferroni test using SPSS 17.0 software package. Difference was considered significant when P value was less than 0.05. Results Overexpressions of MACC1 in ovarian cancer tissues The positive rates of MACC1 in normal ovary, benign ovarian tumor and ovarian cancer tissues were detected by immunohistochemistry (Table 1). Compared to normal ovary and benign ovarian tumor, expressions of MACC1 were obviously up-regulated in ovarian cancer tissues (Figure 1), which showed abnormal expression of MACC1 might be associated with ovarian cancer. Table 1 Expressions of MACC1 protein in different ovarian tissues analyzed by immunohistochemistry.