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Human IFN-�� (0, 1, 2, 5, 20 mM) and/or thimerosal (0, 0.2, 0.5 mM) (Sigma Chemicals, Perth, WA, USA) were added to siRNA-transfected or -untransfected cells. 4. Measurement of cell proliferation After culturing siRNA-transfected (or untransfected) HeLa and C-4I cells with or without thimerosal and/or human IFN-�� for 3 days, supernatants were removed and the wells were washed. For crystal violet assays, 100 mL of 0.5% crystal violet (Sigma Chemicals) solution was added to each well, and then they were incubated for 20 minutes at room temperature. After lysing cells with 100% methanol, optical density was measured at a wavelength of 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). 5. Statistical methods We used the SPSS ver.13.0 (SPSS Inc., Chicago, IL, USA) for the statistical analysis. find more The Mann-Whitney U-test was used to compare differences of cell viabilities after treating targeting siRNAs and/or IFN-�� and thimerosal. Results 1. Decreases in the viability of tumor cells transfected with small interfering RNAs Each siRNA (0, 1, 5, 10, 25, 50 nM) induced a concentration-dependent decrease in the viability of HeLa cells and C-4I cells. The differences in viabilities were appearing from the siRNA concentration of 5 nM and were clearly observed at 50 nM. In HeLa cells, the cell viabilities at siRNA concentration of 50 nM were 44.5% for Sp-18E6/21, 2.5% for Sp-18E6/27, 78.0% for Ex-18E6/21, and 23.5% for Ex-18E6/27 Isoxsuprine (Table 1). Similar results were obtained for C-4I cells; at an siRNA concentration of 50 nM, survival rates were 32.5% for Sp-18E6/21, 2.0% for Sp-18E6/27, 70.5% for Ex-18E6/21, and 22.0% for Ex-18E6/27 (Table 1). Statistical analyses showed significant differences according to each type of siRNAs from the concentration of 5 nM (PSelleck Bcl 2 inhibitor Among four types of siRNAs, the cell viability decreased with the largest scale when Sp-18E6/27 siRNA was transfected. Table 1 The viabilities of HeLa and C-4I cells transfected with different types and concentrations of siRNAs 2. Inhibitory effect of small interfering RNA and interferon beta on HeLa cell growth Sp-18E6/27 siRNA-transfected (10, 25 nM) or -untransfected HeLa cells were incubated with or without IFN-�� collected from MDA-MB-468 and evaluated for the viability. In the absence of added IFN-��, the viability of HeLa cells decreased in proportion to the increase in Sp-18E6/27 siRNA concentration (52.5% at 10 nM and 21.5% at 25 nM). Also, IFN-�� alone induced a rapid and dose-dependent decrease in HeLa cell viability. Notably, as shown in Fig. 1, IFN-�� of low concentrations (1 and 2 mM) in combination with Sp-18E6/27 siRNAs produced the synergistic inhibitory effect to HeLa cell growth. Fig.