KSHV virus production in cells depleted of Rad21.

<p>A. KSHV-infected iSLK cells were transfected with either control siRNA (NC Si) or Rad21-specific siRNA (Rad21 Si), and KSHV replication was induced by treatment with doxycycline. Supernatants from induced cells were used to infect 293 cells. Virus passage was quantitated by flow cytometry of GFP-positive 293 cells. Each transfection/induction was performed in triplicate and three replicate infections were performed with each supernatant. B. CTCF knockdown (CTCF Si) and Rad21 knockdown (Rad21 Si) were performed on iSLK cells in parallel with control siRNA transfection (NC Si). Lytic replication was induced and virus production was measured by passage of virus to KSHV-negative 293 cells as in (A) above. C. CTCF, Rad21 and negative control knockdown were performed in iSLK cells as in (B) above and DNA was isolated from cell pellets. KSHV genome copy number was measured by qPCR. Cells were either untreated (−D) or treated with doxycycline (+D) to induce replication. RQ (relative quantitation). D. Immunoblotting of lysates from cells used in virus production experiments in panel B above was performed with anti-CTCF and anti-Rad21 antibodies to verify completeness of CTCF and Rad21 depletion. Lysates were prepared from cells harvested at the time of replication induction with doxycycline.