Fig 7.

HSV-2-induced apoptosis and maturation block is independent of Akt phosphorylation. (A) DCs were treated with wortmannin (25 μM), rapamycin (0.1 μM), or no drug and were mock infected (M), LPS (10 μg/ml) treated, or infected with HSV-2(G) (MOI = 10 PFU/cell). Cells were harvested 4 h p.i. and analyzed by Western blotting for the expression of p-Akt, total Akt, and β-actin expression. The blot is a representative of two independent experiments. The blots were scanned, the protein levels were quantified, and the percentages of p-Akt relative to total Akt are indicated below each lane. (B and C) In parallel, DCs were challenged with HSV-2(G) (MOI = 5 PFU/cell) in the presence of wortmannin and at 4 h p.i. were analyzed by FACS for apoptosis (B) and maturation (C) (as quantified by CD83 expression). The results are presented as the percent positive for annexin V⁺ 7AAD⁻ (early apoptosis) and annexin V⁺ 7AAD⁺ (late apoptosis) (B) and the fold increase in CD83 expression relative to mock-treated cells (C). The data are means + the SEM of three independent experiments. (D) DCs were treated with wortmannin or no drug and were mock infected or exposed to live or UV-inactivated HSV-2(G) (MOI = 10 PFU/cell) and, at the indicated times postexposure, the cells were lysed and analyzed by Western blotting for the expression of c-FLIP and β-actin. A blot representative of results obtained from two independent experiments is shown, and the percentage of c-FLIP relative to β-actin is indicated below each lane.