Fig 1.

Daxx siRNA knockdown prior to ASV infection results in an initial increase in viral reporter gene expression (GFP) that is reduced over time after restoration of Daxx expression. (A) Experimental outline. Human cells (HeLa, RPE1, and IMR90) were transfected with Daxx siRNA or control siRNA (Dharmacon) and incubated for 96 h. The cells were then infected with the ASV-GFP vector, and GFP expression was analyzed by FACS at 48 h postinfection (p.i.). At the same time, the cultures were sampled to confirm knockdown efficiency by immunoblotting with Daxx antibody. Selected cultures of each cell type were maintained for an additional week, after which time GFP expression was again measured by FACS, and Daxx protein levels were determined by immunoblotting. (B) Assessment of siRNA knockdown of Daxx and its recovery by immunoblotting. Antiactin antibody was used to monitor loading. (C) Daxx knockdown increases ASV-GFP expression over a range of multiplicity of infections (MOIs). Triplicate samples of cells were treated with Daxx siRNA or control siRNA, incubated for 96 h, and infected as described above for panel A. GFP expression was analyzed by FACS at 48 h p.i. MFI, mean fluorescence intensity. (D) Recovery of Daxx protein levels is correlated with repression of ASV-GFP expression. Triplicate samples from the siRNA-treated cells that were either uninfected or infected with 20 μl of viral stock were maintained for an additional week, after which GFP expression was again analyzed by FACS, as described above for panel A. (E) A second treatment with Daxx siRNA resulted in reactivation of GFP. Cell populations originally treated with either control or Daxx siRNAs prior to infection were maintained for 2 weeks as described above for panel D (see the black arrows labeled a and b) and were treated a second time with either Daxx or control siRNAs. GFP expression was analyzed by FACS after 96 h.