br The PC and PC
The PC-3 and PC-3-luc cells were cultured at 2 £ 105 cells/well in 12-well cell culture plates, and cultured at 37˚ C and in 5% CO2 for 24 hours. The PC-3 and PC-3-luc cells were harvested and washed 3 times with phosphate-buff-ered saline (PBS). The PC-3 and PC-3-luc cells were resus-pended in 5 ml 75% ethanol (precooled at 4˚C) and incubated at 4˚C for 18 hours in the dark. Subsequently, the PC-3 and PC-3-luc cells were washed 3 times with PBS and then 500 ml propidium iodide (PI)/RNase solution was added. After 20 minutes of incubation, the sample was transferred to a labeled flow tube and detected using flow cytometry (BD Bioscience Clontech, San Diego, CA).
105 cells/well in 6-well cell culture plates, and cultured at 37˚C and in 5% CO2 for 24 hours. The PC-3 and PC-3-luc cells were collected by centrifugation. Cell lysates were used to resuspend the cells, then they were stored on ice bath for 5 minutes. They were centrifuged at 600 g for 5 minutes and the supernatant was centrifuged obtain both the cytosol and the mitochondria. The precipitants were dis-solved by buffer (10 mmol/L Tris [pH 7.4], 150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA [pH 8.0]); all samples were analyzed by Western blotting. The following primary AZD2281 were used: rabbit b-actin (Cat. 4970), rabbit anti-Cyclin D1 (Cat. 2978), rabbit anti-CDK2 (Cat. 2546) (all from Cell signaling technology, Danvers MA). The following secondary antibodies were used: Peroxidase-Conjugated Goat anti-Rabbit IgG (H+L) (Cat. ZB2301, ZSGB-BIO, CN).
2.4. Crystal violet staining assay
Cell survival was measured using crystal violet staining to observe the inhibition of recombinant adenovirus on the growth of the PC-3-luc cells. The PC-3-luc cells were cul-tured at 2 £ 105 cells/well in 12-well cell culture plates, and cultured at 37˚C and in 5% CO2 for 24 hours. Subse-quently, the PC-3-luc cells were infected with different concentrations of recombinant adenovirus (1 multiplicity of infection [MOI], 10 MOI, and 100 MOI). At 24, 48, 72, and 96 hours, the culture medium in each well was discarded
and the plates were turned over on a filter paper for 1 min-ute, and then 350 ml of 0.1% crystal violet solution were added to each well for the staining at room temperature for 10 minutes. The staining solution was carefully sucked dry by turning the plates over on a filter paper. The inhibitory effects of the recombinant adenovirus on the growth of the PC-3-luc cells were then observed.
PC-3-luc cells were cultured at 2 £ 105 cells/well in 12-well cell culture plates, and cultured at 37˚C and in 5% CO2 for 24 hours. Subsequently, the cells were infected with different concentrations of recombinant adenovirus (1 MOI, 10 MOI, and 100 MOI). At 24, 48, 72, and 96 hours, the PC-3-luc cells were harvested and washed 3 times with PBS. PC-3-luc cells were resuspended in 50 ml PBS and then 1 ml of the Hoechst solution was added. After 15 minutes of incubation, a 10 ml sample was applied to a microscope slide with a cover slip, and then observed and photographed under a fluorescence microscope (BX-60, Olympus, Tokyo, Japan). Tests were performed in triplicate and at least 500 cells were scored for each sample to deter-mine the nuclear changes.
2.7. Annexin V analysis
PC-3-luc cells were infected with the recombinant adenoviruses (100 MOI). After 48 hours, the cells (2 £ 105) were harvested and resuspended in binding buffer and stained with fluorescein isothiocyanate (FITC)-labeled Annexin V (Annexin V-FITC Apoptosis Detection Kit; BioVision, Mountain View, CA) according to the man-ufacturer’s protocols. To exclude late apoptotic and necrotic cells, PI was added to the FITC-Annexin V-stained samples. The samples were then examined by flow cytome-try (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ) for apoptosis analysis (Cell Quest Pro, Becton Dickinson).