(A) Knock-down of cellular DNA ligases. HeLa cells were transfected with siRNAs to Lig1, 3 or 4 or a control non-targeting RNA. After 72 h, cell lysates were analyzed by Western blotting with antibodies to Lig1, 3, 4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a loading control. Antibody binding was detected by chemiluminescence. (B) Effects of siRNAs on replication of mutant and wild type VACV. HeLa cells were transfected with siRNAs to Lig1, 3, or 4 or with a control non-targeting siRNA. After 72 h, the cells were infected with 4 PFU per cell of vΔA50gfp or vA50Rev. At indicated times, virus titers were determined by plaque assay (upper panels) or viral DNA was quantified by real-time PCR (lower panels). Experiments were in triplicate and bars represent standard error of the mean. (C) Effect of Lig1 knock-down on late gene expression. HeLa cells were transfected with ligase-specific or control siRNA. After 72 h, the cells were infected with 4 PFU per cell of vΔA50gfp and visualized by fluorescence microscopy at indicated times.