Figure 9. Depletion of ND10 components does not interfere with latency establishment in SLK or EA.hy cells.

Cell lines depleted for Sp100, Daxx, PML or GFP (control) were generated by transduction with lentiviruses expressing specific shRNAs. After antibiotic selection, stable SLK (A) and EA.hy (B) cultures were analyzed by western blotting to confirm successful knock down. (C): FACS analysis to establish the frequency of ORF59 positive cells in SLK knockdown cultures. The right panel shows a positive staining control employing TPA (20 nM) and sodium butyrate (0.3 mM) induced) BCBL1 cultures 48 h after treatment. Columns 1 to 4 of the left panel show SLK knockdown cultures at 36 h of infection with KSHV. As an additional positive control for ORF59 staining, the rightmost column of the panel shows lytically reactivated cells from long-term infected and overconfluently grown SLKp cultures (see text for details). (D): FACS analysis of stably shRNA-expressing EA.hy cultures analysed at 48 hours post infection with KSHV. The rightmost columns show cells which were treated with sodium butyrate (2.5 mM) immediately after infection. Mock infected cells were used to correct for background staining levels in all experiments. Error bars represent SEM of at least two and up to four biological replicates. (E) Transcript levels of ORF50, ORF59, ORF64 and ORF73 in EA.hy cells at 48 hours post infection (see Table S1 for RT-qPCR primers). Expression was calculated by normalization to GAPDH and is shown relative to shGFP controls (set to 1). Error bars represent SEM of at least three data sets.