Fig. 3

HSV-1 induces necroptotic cell death in primary astrocytes by both a RIPK1-independent ZBP1-mediated pathway and a RIPK1-mediated ZBP1-independent pathway. ZBP1+/+ and ZBP1−/− murine astrocytes were infected with HSV-1(MacIntyre), HSV-1(F)-ICP6-RHIM Mut, or its parental ICP6-expressing parental strain (HSV-1(F)). One hour following infection, cells were treated with either DMSO vehicle control (A, B) or the RIPK1 inhibitor GSK963 (1 μM) (C, D). A, C Cell viability was measured every two hours with a RealTime-Glo™ MT assay beginning at two hours following infection. B, D At 24 h, total cell lysates were collected and analyzed for the presence of phosphorylated MLKL (P-MLKL) or the housekeeping gene product β-actin (Actin) by immunoblot analysis. Relative P-MLKL expression was determined by densitometric analysis and normalized to β-actin expression levels. Data are shown as the mean of three independent experiments ± SEM. An asterisk indicates a significant difference in final cell death or P-MLKL expression from similarly treated ZBP1+/+ cells and dagger symbols indicate a significant difference from uninfected cells