FIG 2.

Both knockdown of Ikaros and expression of a dominant-negative isoform, IK-6, enhance lytic EBV reactivation. (A) Immunoblots showing relative levels of some lytic EBV-encoded proteins following shRNA knockdown of Ikaros and incubation without (−) or with (+) TGF-β1. Sal and MutuI cells were infected for 3 days with lentivirus expressing nontargeting shRNA (Control #1) or a combination of five shRNAs targeting Ikaros, incubated for 4 days in the presence of puromycin (1 μg/ml), and then incubated for 24 h in the absence or presence of TGF-β1 (100 pM) immediately prior to preparing whole-cell extracts. (B) Immunoblots showing lytic EBV proteins following superinfection of Sal cells expressing the indicated shRNAs with lentivirus expressing IK-1 and incubation with TGF-β1. Cells were infected for 24 h with lentiviruses expressing nontargeting shRNAs (Control #1 and Control #2) or a combination of four shRNAs targeting Ikaros, superinfected for 2 days with 525 lentivirus expressing IK-1 (IK-1) or 525 as empty vector (Control), selected for 5 days with puromycin, and then incubated for 24 h with TGF-β1. (C) Immunoblots showing lytic EBV proteins following infection of Sal cells for 3 days with lentiviruses expressing the indicated isoforms of Ikaros, followed by incubation for 24 h with 0.2 mM hypoxia mimic DFO (+) or with DMSO as a control (−). (D) Immunoblots showing lytic EBV proteins following infection of MutuI cells for 3 days with lentiviruses expressing the indicated isoforms of Ikaros and incubation for 24 h with TGF-β1.