Hydrogen peroxide leads to rapid loss of the cell membrane integrity in nef -expressing astrocytes. (A) U251MG-parental, -pNeo and -Nef cells were treated with hydrogen peroxide for 30 min at concentrations as indicated. Cells then were washed twice with PBS, incubated in VLE-RPMI 1640 medium containing 10% FCS for a further 3 h 30 min and subsequently the annexin V assay was performed as described in the Methods section. (B) Summary of three independent experiments. Annexin V, PI double-negative cells (living cells) and annexin V, PI double-positive cells (dead cells) are shown.