Figure 2
Vpr and C81 induce apoptosis in tumor cells. (A) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag. At 48 h post-transfection, cells were fixed and stained with anti-Flag, followed by Alexa-488-conjugated anti-mouse IgG. Finally, cells were stained with Hoechst 33258 to monitor the nuclear morphology. Apoptotic bodies (arrowheads) were identified using confocal laser scanning microscopy. (B) HeLa, HepG2, and 293 T cells were transfected with the pME18Neo plasmid encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag, together with pSV-β-galactosidase. Cells were treated with (filled columns) or without (open columns) inhibitors of caspase-3. At 30 h (293 T), 36 h (HeLa), or 48 h (HepG2) post-transfection, caspase-3 activity was measured and normalized to the β-galactosidase activity. Each column and error bar represents the mean ± SD of measurements from three samples. (C) HeLa, HepG2, and 293 T cells were transfected with pME18Neo encoding Flag-tagged wild-type Vpr or C81, or the control pME18Neo-Flag with or without the GFP expression vector pEGFP-N1. GFP was used as a reporter to discriminate between transfected and untransfected cells. At 47 h (293 T), 38 h (HeLa), or 58 h (HepG2) post-transfection, cells were stained with PE-Annexin V and 7-AAD to identify apoptotic cells, or anti-mouse IgG2b-PE (Nippon BD Company Ltd) and 7-AAD as a negative control. The percentages of Annexin V-positive and 7-AAD negative cells relative to GFP-positive cells indicate the level of Vpr or C81 associated apoptosis.