FIGURE 4

Effects of CSE on lung cancer cells proliferation and invasion. (a) A549 cells were seeded in 24-well culture plates at a density of 1 10⁴ cells/well and cultured in 0.5 ml medium containing 10% FBS without or with 5% CSE. At indicated time, cells were trypsinized and viable cells were counted. Values are the mean of triplicate wells. (b) A549 cells were treated with 0, 1.2 and 5% CSE for 3 days. The invasive capacities of cells were determined using two-compartment Boyden chambers and the basement membrane matrigel. The 8 m pore polycarbonate filters were coated with matrigel and used to analyse 5 10⁴ cells in each chamber. After 40 h, filters were fixed, washed and stained. Cells were examined under a light microscope. Under 200 magnification, 10 randomly selected fields were examined and the average number of cells invaded per field was calculated. Photographs of representative views from control and CSE-treated cells are shown in the top panel and the numbers of invaded cells are presented in the bottom graph. The data shown are representative of three separate experiments. ^** P<0.01 as compared to untreated control. (c) siRNAs directed to two different regions of SNCG mRNA were cotransfected into A549 cells. A nonspecific siRNA with scrambled sequence was also transfected into A549 cells as transfection control. Twenty-four hour posttransfection, cells were treated with 5% CSE for 3 days. siRNA-transfected cells and nontransfected cells were trypsinized and were tested in Boyden chamber assay for their invasive capacities. ^* P<0.05 as compared to without siRNA. (d) H292 cells were transfected with SNCG siRNA or the scrambled control siRNA for 3 days. A portion of transfected cells was used in Boyden chamber assay and rest of cells were lysed for Western blot analysis of SNCG protein expression. ^* P<0.05 as compared to without siRNA.