Fig 3. HIF-1α addition is sufficient to induce EBV reactivation and necessary for efficient induction by DFO.

(A) Immunoblots showing addition of HIF-1α is sufficient to induce lytic EBV reactivation. AGS-Akata cells grown in 10-cm dishes were transfected with 1 μg each of pHA-HIF-1α P402A/P564A-pcDNA3 plus pHIF-β (+) or 2 μg of their empty vector, pcDNA3, as a control (-) and incubated for 48 h prior to preparation of whole-cell extracts. Data are representative of numerous independent experiments. (B) Immunoblots showing knockdown of HIF-1α inhibits DFO-induced synthesis of EBV lytic antigens. Lanes 1–6, AGS-Akata cells maintained in 10-cm dishes were co-transfected with 0.8 μg of each of five lentiviruses that express different shRNAs targeting HIF-1α (lanes 5–6) or 4 μg of a lentivirus that expresses the non-targeting shRNA cntl. #1 or cntl. #2 (lanes 1–2 and lanes 3–4, respectively). Two days later, the cells were incubated in the absence (-) or presence (+) of 200 μM DFO for 24 h prior to harvesting and preparation of whole-cell extracts. Lanes 7–10, Sal cells were infected with the indicated packaged lentiviruses; three days later, the cells were incubated in the absence (-) or presence (+) of 200 μM DFO for 24 h and processed likewise. Data are representative of two independent experiments. GADPH served as a loading control.