As a control, cells were transfected with the individual siRNAs at a concentration of 10 nM. To correct for potential saturation effects (e.g., during transfection and/or RISC loading of siRNAs), Selleckchem PD-1/PD-L1 inhibitor cells were also transfected with a combination of 5 nM individual targeting siRNA and 5 nM non-targeting control siRNA. The numbers of infectious virus particles were determined at 48 h post-infection

by TCID50 assay ( Fig. 7). As shown in Fig. 7B, the superior anti-adenoviral effect mediated by the DNA polymerase siRNA was not enhanced by simultaneous targeting of those mRNAs whose generation depends on the function of the DNA polymerase, e.g., the IVa2 or hexon genes. Similarly, combined E1A and DNA polymerase silencing did not further decrease virus titers ( Fig. 7A). The same held true for all other siRNA combinations. In general, combining a highly effective siRNA with a less well-performing siRNA led to an intermediate inhibition rate, or an inhibition rate equal to the one caused by the individual better-performing siRNA. Moreover, the anti-adenoviral effect

of an individual siRNA was not reduced by SB431542 in vivo halving its concentration upon combination with an equal concentration of non-targeting negative control siRNA. We speculated that possible synergistic effects may have been undetectable, because the cells were harvested at a relatively early time point (48 h post-infection). However, they might become detectable at later time points, when the virus was allowed to spread throughout the culture. We hypothesized that combinations comprising the E1A siRNA on the one hand, and siRNAs targeting mRNAs

originating from other early/middle genes on the other, would be most likely to cause a synergistic effect. We therefore repeated the virus inhibition experiment using the respective siRNA combinations, and determined Ad5 genome copy numbers at 6 days post-infection. However, we did not detect any synergistic effects at this late time point (Supplementary Fig. 4). We also repeated the experiment using lower concentrations of siRNAs. Although there was a slight trend toward somewhat increased inhibition for some combinations, none of these differences were statistically significant, and under no conditions did any combinations of siRNAs result in a higher inhibition Terminal deoxynucleotidyl transferase rate than the inhibition rate caused by Pol-si2 when applied alone (Supplementary Fig. 5). Next, we quantitatively assessed the impact of Ad5 gene silencing on the viability of infected cultures. We transfected A549 cells with the siRNAs at a concentration of 10 nM as before, and then infected them with Ad5 at a higher MOI (4 TCID50/cell) to ensure pronounced cell killing. We determined the metabolic activity as a measure of cell viability at 6 days post-infection, by means of an MTS assay (Fig. 8). As expected, the siRNAs, although greatly decreasing the output of virus progeny, were not capable of preventing already infected cells from cell death.