Hydrogen peroxide decreased the viability of nef -expressing astrocytes. U251MG-parental, -pNeo and -Nef cells were treated with hydrogen peroxide for 30 min at indicated concentrations. Subsequently cells were washed twice with PBS and incubated in VLE-RPMI 1640 medium containing 10% FCS for a further 24 h. The medium was then exchanged and cell viability assay was performed as described in the Material section. The times indicated are relative to the moment of adding AlamarBlue® reagent to the cell culture medium. The relative fluorescence represents the ratio of the fluorescence intensity of study cells versus mock-treated cells at 1 h after start of the assay. Data represent mean ± s.e.m. (n = 6); **, P < 0.01.