After transfection, 786-O cells were starved in serum free medium overnight, and 3-5 × 104 cells were resuspended in 200 ul serum-free medium and placed in the upper chambers with 8 μm filter pores in triplicate. The membrane undersurface was coated with 30 ul ECM gel from Engelbreth-Holm-Swarm mouse sarcoma (BD Biosciences, Bedford, MA, USA) mixed with RPMI-1640 www.selleckchem.com/products/Vorinostat-saha.html serum free medium in 1:5 dilution for 30 min at 37°. The lower chamber was filled with 500 ul 10% FBS as the chemoattractant and check details incubated for 48 h. At
the end of the experiments, the cells on the upper surface of the membrane were removed by cotton buds, and the cells on the lower surfaPBS-buffered paraformaldehyde and stained with 0.1% crystal violet. Five visual fields were chosen randomly for each insert and photographed under a light microscope at 200 × magnification. The cells were counted and the data were summarized by means ± standard deviation and presented by a percentage of controls. ce of the insert were fixed in 4%. Gelatin zymography assay After transfection, the cells were cultured in serum free medium for 24 h. Then the medium was collected by centrifugation at 4,000 rpm Metabolism inhibitor for 15 min at 4°C, and subjected to zymographic SDS-PAGE containing 0.1% gelatin (w/v). The gels were washed and incubated in incubation buffer for 48 h, then stained with Coomassie Brilliant Blue and destained.
The zones of gelatinolytic activity were shown by negative staining.
Tumourigenesis assay in nude mice Female BALB/cnu/numice (4-6 weeks old, weighed 25-30 g) were maintained BCKDHA in a germ-free environment in the animal facility. NSBP1 knockdown or control 786-O cells were cultured in 100-mm dishes and trypsinized. The cells (10 6 in 100 ul medium) were infused subcutaneously in the armpit area. Tumor diameter was measured every 5 days, and tumor volume was calculated by length × width2× 0.5. Mice were sacrificed after 1.5 months. Statistical analysis Values were represented as mean ± SD for at least triplicate determination, and analyzed using Fisher’s exact test and Kruskal-Wallis test. All statistical analyses were performed using SPSS 13.0 and P < 0.05 was considered as statistically significant. Results NSBP1 expression is high in ccRCC tissues We examined NSBP1 expression in ccRCC tissue by immunohistochemistry. As shown in Figure 1A, NSBP1 staining was weak in the normal renal tissues but strong in ccRCC tissues. Western blot analysis of 20 paired adjacent normal renal tissues and ccRCC tissues confirmed the high expression of NSBP1 in ccRCC tissues (p = 0.006) (Figure 1B). Most importantly, we found that NSBP1 staining intensity was correlated with the clinical and pathologic characteristics of ccRCC (Table 1). NSBP1 expression was positively correlated with the tumor grade and pathologic stage. Figure 1 NSBP1 expression is high in ccRCC tissues and cells.