(A) Tyrosine phosphorylation of STAT3 in KSHV⁺ PEL cells.

Cell lysates (2 × 10⁶ cell equivalents per lane) from BAF130 cells (positive control), K562 cells (negative control), and PEL cell lines were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. BAF130 cells were stimulated with 1 μg/mL MBP–vIL-6 at 37°C for 10 minutes before lysis. Phosphorylated STAT3 was visualized with the use of phospho-specific Ab recognizing Tyr705-phosphorylated STAT3. The same filter was stripped and blotted with an Ab for unphosphorylated STAT3, followed by restripping and reblotting with an Ab to α-actin. Data shown are representative of 3 independent experiments. (B) EGFP-STAT3D–expressing and control EGFP-expressing vectors were transfected into PEL and K562 cells. At 3 days after transfection, cells were stained with annexin V–PE and counted under a fluorescent microscope. The percentage of apoptosis indicates the fraction of annexin V–PE⁺ population within EGFP-expressing cells. Data represent the mean (± standard deviation [SD]) of 3 experiments. (C) EGFP⁺ VG-1 cells were sorted 2 days after transfection, and cultured in fresh medium for 1 day, followed by staining with annexin V–PE. Most cells expressing EGFP-STAT3D were positive for annexin V. Original magnification × 320. (D) At 2 and 3 days after transfection, VG-1 and K562 cells were stained with PI and analyzed by flow cytometry. A minimum of 5000 EGFP⁺ cells were analyzed for each sample. Shown is a representative of 4 experiments performed. FSC indicates forward scatter.