Fig. 4

The notion that ZBP1 mediates multiple cell death pathways in HSV-1 challenged primary murine astrocytes is supported by similar results obtained using alternative pharmacological inhibitors. ZBP1+/+ and ZBP1−/− murine astrocytes were infected with HSV-1(MacIntyre) (A), or HSV-1(F)-ICP6-RHIM Mut or its parental ICP6-expressing parental strain (HSV-1(F)) (B). One hour following infection, cells were treated with either DMSO vehicle control, the RIPK1 inhibitor GSK547 (50 nM), the RIPK3 inhibitor GSK843 (2 μM), and/or the caspase-8 inhibitor Z-IETD-FMK (20 μM). A Cell viability was measured at 24 h following infection with a RealTime-Glo™ MT assay. Panel B: At 24 h, total cell lysates were collected and analyzed for the presence of phosphorylated MLKL (P-MLKL). Relative P-MLKL expression determined by densitometric analysis is shown 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 untreated infected cells