FIG. 3.
Induction of A20-HE cell reactivation. (A) Immunoblot analysis for lytic cycle-associated antigens following B-cell-activating stimulation. A total of 5 × 105 A20-HE cells per ml were treated as follows: 10 μg/ml LPS, 20 ng/ml PMA, 2 mM NaB, 5 μg/ml anti-immunoglobulin G (α-Ig), 2.5 μg/ml anti-CD40, or the combinations indicated at the same concentrations. Mock-infected or infected (MOI, 5 PFU/cell, 24 h postinfection) NIH 3T3 fibroblasts served as respective positive and negative controls. Cells were harvested 24 h after treatment. Forty μg of total protein for A20-HE cell samples (12.5 μg for 3T3s) was resolved by SDS-PAGE followed by immunoblot analysis for the indicated proteins. The nonspecific band was included as a loading control for A20-HE samples. (B) Immunofluorescence analysis following stimulation. A20-HE cells were treated with the indicated stimuli as in panel A and processed for immunofluorescence microscopy 24 h after treatment. Cells were stained with antibodies to GFP and MHV68 antisera. DNA was detected with DAPI in the mounting medium. Magnification, ×100. (C) Virus production following stimulation. A20-HE cells were treated with the indicated stimuli as for panel A. Cells were harvested at the indicated times posttreatment, and viral titers were determined by plaque assay. Data represent the fold increase in titer following treatment relative to mock-treated controls. Results are means of triplicate samples. Error bars represent standard deviations.