Targeting LANA with shRNA. (A) Western blot shows that after infection with sh-LANA, knock-down remains stable in BC-3 cells in puromycin selection for at least 6 months. (B) MTT assay for cell proliferation showing no significant difference between empty vector and sh-LANA cells 6 months after infection. The horizontal bar indicates the mean value. (C) BrdU assay showing no significant difference in 5-bromo-2′-deoxyuridine uptake between empty vector and sh-LANA cells 6 months after infection. The horizontal bar indicates the mean value. (D) qPCR for LANA DNA as a representation of KSHV DNA copy number shows that KSHV copy number gradually decreases in BC-3 cells until it reaches a plateau. (E) Graph of cell doubling time in KSHV negative B-cell (Ramos) and KSHV-positive PEL cell lines (BC-3, JSC-1, and BCP-1), 2 or 7 days after infection with empty vector (▦, day 2; ▩ , day 7) or with sh-LANA (▨, day 2; ▧, day 7). □ represents untreated cells. LANA knock-down has different effects on different lines. (F) Graph shows effect of increasing MOI (in 293t IU/cell) with sh-LANA on doubling time of JSC-1 cells 10 days after infection. Western blot shows LANA levels in these cells. (G) qRT-PCR for ORF50 (Rta) mRNA as an indicator of activation of lytic replication in JSC-1 cells before and after infection with sh-LANA and with or without the addition of TPA to induce lytic replication. Error bars represent the standard error of the mean.