The cells were added to the upper chamber at a density of 4 × ATM Kinase Inhibitor in vitro 104 cells/insert, After 24 h of incubation, cells on the upper surface were wiped off with a cotton swab. Cells that had invaded the lower surface were fixed with 70% ethanol, stained with 0.2% crystal violet, Invasiveness was quantitated by selecting ten different views (100 times) and calculating the number of invading cells. Gilteritinib chemical structure Migration assay Migration assays were performed using two-chamber-Transwell (Corning, USA) as described previously

[20]. The lower surface of a polycarbonate filter with 8 μm pores was coated with 1 μg/ml bovine collagen IV. Cells were trypsinized and suspended in a serum-free medium containing 1% BSA at a concentration of 4 × 104 cells/insert. The cells were placed in the upper chamber and free DMEM was placed in the lower chamber. After 12 hr at 37°C, the cells in the upper chamber were wiped off with a cotton swab. The cells on the lower surface of the filter were fixed with 70% ethanol, stained with 0.2% crystal violet, migration was quantitated by selecting ten different views (100 times) and calculating the number of migrated cells. Statistical analysis All statistical analyses were performed using SPSS

10.0. VX-765 mw Data were expressed as mean ± SD. The statistical correlation of data between groups was analyzed by one-way analysis of variance (ANOVA) and Student’s t test, where P < 0.05 were considered significant. Results

Selection of the most effective COX-2 specific shRNA expression vector To exclude off-target Temsirolimus silencing effects mediated by specific shRNA, we employed three different COX-2 shRNAs (shRNA1, shRNA2, shRNA3). Three specific plasmids and the control plasmid were cotransfected with packing plasmid into 293T cells, respectively. 48 h after transfection, GFP expression in 293T cells was observed under a fluorescent microscope (Figure 1a). The level of COX-2 expression was evaluated by RT-PCR and western blotting. Results indicated that all of the COX-2shRNA-1, shRNA-2 and shRNA-3 significantly decreased the COX-2 mRNA and protein levels in 293T cells. According to the results, LV-COX-2siRNA-1 was the most effective lentivirus vector, and was used in the following experiments (Figure 1b and 1c). Figure 1 Downregulation of COX-2 expression in 293T cells by shRNA transfection. (A) GFP expressed 48 h after the transfection of the control, shRNA1, shRNA2 and shRNA3 plasmid in 293T cells, under a fluorescent microscope, respectively. (magnification 200 ×). (B) COX-2 mRNA levels were detected by RT-PCR. (C) COX-2 protein levels were detected by western blotting. Data are presented as mean ± s.e.m. * P < 0.01, # P < 0.001, compared with untransfected 293T cells group or control plasmid transfected cells group.