Figure 2: CCTα siRNA sequences induce A549 cell death. A549 cells cultured to 80% confluence were transfected with siRNA S and CCTα siRNA A, B and C (0–800 nM), preincubated with FuGENE 6 at room temperature as indicated above. After 24 hr incubation, cells were washed three times with PBS and incubated with Serum free media. Cell images were visualized and captured by an Olympus fluorescent microscope (40X objective) fitted with an Olympus DP70 camera. Images were documented using Adobe Photoshop. To determine cell viability, A549 cells were treated with different concentrations of CCTα-siRNAs in 96 well plates and living or dead cells detected by cell titre blue (CTB) or lactate dehydrogenase (LDH) cell viability assays, respectively. Briefly, cells treated with CCTα siRNAs in serum free media as indicated above were grown for 24 h, 20μl CTB reagent were afterwards added to the cells in each well. After a 1.5 h incubation, fluorescence was read at excitation/ emission wavelengths of 560/590 nm in a Microplate Foursecence Reader FLx 800. For LDH assay, 50μl of supernatant obtained from cells grown under identical conditions as described in CTB assay were transferred from each well to a new 96 well plate. 50μl of LDH detection solution added to supernatants. After 30 min incubation at room temperature in the dark, absorbance was read at a wavelength of 495 nm in a POWER X 340. As indicated in Figures 2A (Light microscopy), B (CTB) and C (LDH), CCTα siRNA induced cell death in A549 cells. Untreated and siRNA S treated cells remained intact and exhibited no significant differences (p > 0.05) in the fluorescence intensity and absorbance, for CTB and LDH assay, respectively.