The supernatant containing the cell mem brane proteins was stored at 80 C until use. Protein quantification was performed using the BCA kit according to the instructions http://www.selleckchem.com/products/MG132.html of the manu facturer. Total protein was electrophoresed Inhibitors,Modulators,Libraries using a PAC300 electrophoresis instrument according to the instructions of the manufacturer. Proteins were then transferred to a cellulose nitrate membrane using an electrophoretic Inhibitors,Modulators,Libraries transfer instrument. Western blot analyses were performed and the bands visualized using an ECL kit. All the required antibodies were purchased from Santa Cruz Biotechnol ogy. Films were developed using an X XQF 2000 gel imaging system. Quantitation was done by estimating the optical density of the bands. The actin band was used as a normalizing control.
MMP 9 and uPA immunofluorescent staining Cells were inoculated into culture wells with a strip shaped cover slip, incubated for 48 h and rinsed with PBS twice. The cells were fixed with cold acetone for 10 min and stored at 20 C until use. Immunofluorescent stain Inhibitors,Modulators,Libraries ing was performed as follows. The cell climbing slices were soaked in 1% Triton TBS buffer for 30 min and rinsed with PBS. Samples were blocked with non immune animal serum at 37 C for 15 20 min and incubated in the diluted first antibody at 4 C overnight. The samples were rinsed with PBS and then incubated in the dark with second antibody labeled with the corresponding fluores cein moiety at 37 C for 2 h. Samples were rinsed with PBS and observed under a Leica DM LB2 fluorescence micro scope. For the negative control, the first antibody was replaced by PBS and the remaining procedures and reagents were unchanged.
The appearance of red fluorescent areas in the cytoplasm was associated with the presence of MMP 9. the appearance of green flu orescent areas in the cytoplasm was associated with the presence of uPA. Intracellular protein levels were meas ured using the automatic image analysis Inhibitors,Modulators,Libraries instrument of the microscope system. Using the 200�� magnified visual field, 100 cells were randomly selected from the upper, lower, left, right, and central zones of the slides in the same experiment. The average optical density value of the fluorescent particles was measured and the measurement was repeated four times. The data were shown in mean standard deviation. Statistical analysis The experimental data are shown in mean standard deviation.
Inhibitors,Modulators,Libraries The one way ANOVA was used for the comparisons among the means. All the analyses were car ried out using the SPSS13. 0 software. The significance level was set at P 0. 05. Results Effects of staurosporine on the cell morphology Electron microscopy analyses demonstrated that cell assay untreated A549 cells were short spindle shaped and trian gle shaped. They exhibited characteristics resembling those of epithelial cells. Upon treatment with 1 nmol L staurosporine for 24 h, morphological changes were observed.