Figure 7.

Effects of ROS-modulating agents on HSV-1 replication. U937 cells were pre-incubated with selenium-based antioxidants (SeDA4 or SeMC 20 µM) for 48 h and/or exposed to H₂O₂ for a further 30 min before infection with HSV-1 (MOI 50 PFU/cell). At the indicated times, cells (hpi) were collected and assayed for virus replication by different techniques. (A,B) Western blot analysis of ICPO (A) and gD (B) viral proteins from samples pre-incubated with antioxidants or complete media (vehicle) before exposure to H₂O₂ and infection. Mock-infected cultures were also included. Top panels: densitometric analysis normalized to β-actin expression and expressed as mean + S.D of data obtained from two evaluations in duplicates. Bottom panels: representative gels. (C) Immunofluorescence microscopy analysis of gD expressing cells from samples pre-incubated with antioxidants or complete medium (vehicle) before exposure to H₂O₂ and infection. Analysis by IFA is expressed as % positive cells (mean + S.D.) calculated from 20 different fields per conditions of four separate experiments (5 fields each). (D) Virus yield in supernatants determined by plaque assay in samples obtained from the same experiments of C. Data are expressed as percentage of HSV-1 yield with respect to infected, untreated cells (vehicle). * p < 0.05, ** p < 0.001, *** p < 0.0001 versus HSV-1-vehicle treated groups (only infected groups are represented).