FIG. 4.

Ectopic expression of Wee-1 inhibits Vpr-induced apoptosis. Wee-1 with a C-terminal HA tag was expressed with a lentiviral vector derived from pHR′CMV-EGFP (1). HR′Wee1 and HR′Thy (Vpr⁺) viruses were made as described in the text. HR′Thy (Vpr⁺) packages Vpr but does not express Vpr de novo (30). HR′Thy (Vpr⁺) was titrated as described in the legend for Fig. 1. A virus inoculum equivalent to 2.5 μg of p24 was needed to achieve more than 95% G₂ arrest in 2 × 10⁵ HeLa cells. Cells were infected by HR′Wee-1 or HR′Thy (control) 1 day before HR′Thy (Vpr⁺) or HR′Thy (control) infection. Viruses with an equivalent amount of p24 were used in each infection. Cells were stained either with Annexin V and 7-AAD or with PI or were lysed for Western blotting 72 h postinfection. (A) Expression of HA-tagged Wee-1 in the coinfected HeLa cells. Cell lysates were fractionated by SDS-10% PAGE and immunoblotted with HA antibody (Convence) as described in the legend for Fig. 1. (B) Cell cycle analysis of coinfected HeLa cells. (C) Apoptosis of HeLa cells coinfected with viruses as indicated.