FIG 6.

MHV68-infected cells become nonresponsive to p53 agonists as infection progresses. (A) 3T3 fibroblasts were mock infected or infected with wild-type MHV68 at an MOI of 5 PFU/cell. Cells were treated with vehicle (DMSO) or doxorubicin (5 μM) 4 h prior to harvest at the indicated times postinfection. Cells were harvested in RIPA buffer at the indicated times postinfection, and lysates were resolved by SDS-PAGE. Resolved proteins were detected by immunoblot analyses using antibodies that recognize the indicated proteins. Detection of β-actin serves as a loading control. (B and C) 3T3 fibroblasts were infected with MHV68 at an MOI of 0.5 PFU/cell. Cells were treated with vehicle or doxorubicin (5 μM) 14 h postinfection. Cells were fixed with 10% formalin 18 h postinfection and processed for immunofluorescence microscopy. Processed cells were stained to detect viral antigen ORF59 and p53. DNA was detected with DAPI. (C) Relative fluorescent intensities of p53 in ORF59-negative or ORF59-positive cells from panel B were quantified in multiple images using Nikon NIS Elements software. Data represent relative p53 fluorescence intensity from 32 randomly selected cells for each condition.