FIG. 8.

Verification of the generation of double-knockdown HFFs (siDaxx1+siPML2) devoid of both ND10 constituents, PML and hDaxx, followed by the analysis of HCMV replication in the constructed double-knockdown cells compared to single-knockdown fibroblasts. (A) Western blotting for detection of hDaxx and PML expression in the generated double-knockdown cells, siDaxx1+siPML2. Comparison of PML (upper panel), hDaxx (middle panel), and actin protein levels (lower panel) in whole-cell extracts from double-knockdown (siDaxx1+siPML2), single-knockdown (siPML2, siDaxx1), and control HFFs (vector and siC). (B) Analysis of HCMV IE gene expression after infection of double-knockdown cells in comparison to single-knockdown fibroblasts. The different cell populations as indicated were grown on coverslips in six-well dishes and infected with 100 IEU/well of wt HCMV AD169. At 24 hpi the amount of IE1-expressing cells was assessed via indirect immunofluorescence analysis. Error bars illustrate the standard deviation of three independent determinations.