FIG 1.

Reovirus-induces necroptosis in L929 cells. (A) L929 cells infected with 10 PFU/cell of T3D for 34 h were fixed, stained, and imaged by using transmission electron microscopy. (B) Cell death in L929 cells 48 h following mock infection or infection with 10 PFU/cell of T3D and treatment with DMSO or Q-VD-OPh (20 μM) was assessed by using the Cell Titer Glo system. Luminescence measurement in similarly treated, uninfected cells was considered to represent 100% viability. (C) Caspase-3/7 activity 48 h following infection of L929 cells with 10 PFU/cell of T3D and treatment with DMSO or Q-VD-OPh was assessed by a chemiluminescent enzymatic assay. The value for caspase activity in mock-infected cells was set to 1. Data are presented as relative caspase-3/7 activity in comparison to the activity in similarly treated, uninfected cells. *, P < 0.05 compared to cells treated with DMSO. (D to G) L929 cells were transfected with nontargeting siRNAs or siRNAs specific for RIP3. (D) Efficiency of knockdown was assessed by immunoblotting for RIP3 and the PSTAIR loading control. (E) Cell death 48 h following mock infection or infection with 10 PFU/cell of T3D was assessed by using the Cell Titer Glo system. Luminescence measurement in similarly treated, uninfected cells was considered to represent 100% viability. *, P < 0.05 compared to cells transfected with nontargeting siRNAs. (F) Cell death 48 h following infection with 10 PFU/cell of T3D was assessed by AOEB staining. *, P < 0.05 compared to cells transfected with nontargeting siRNAs. EtBr, ethidium bromide. (G) Cell death 3 h following treatment with TNF-α and Z-VAD-FMK was assessed by using the Cell Titer Glo system. Luminescence measurement in similarly siRNA treated, DMSO-treated cells was considered to represent 100% viability. (H) Whole-cell extracts from L929 cells infected with 10 PFU/cell of T3D at the indicated time points were immunoblotted for phosphorylated MLKL, total MLKL, and the PSTAIR loading control.