Effect of CTCF depletion on KSHV virion production.
A. CTCF knockdown in iSLK cells. Cells were transfected with either negative control siRNA (NC Si) or siRNA specific for CTCF (CTCF Si). Cell lysates were prepared 48 h after transfection and analyzed by immunoblotting with an anti-CTCF antibody (upper panel). Blots were stripped and re-probed with an anti-actin antibody as a loading control (lower panel). B. KSHV virus production in cells depleted of CTCF. KSHV-infected iSLK cells were transfected with either control siRNA (NC Si) or CTCF-specific siRNA (CTCF 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. Uninduced cell supernatants from control and CTCF depleted cells contained less than twenty-five detectable infectious particles per ml (data not shown).