Journal: Cell Death and Differentiation
Article Title: Induction of necroptotic cell death by viral activation of the RIG-I or STING pathway
Figure Lengend Snippet: Sendai virus Y1/Y2 proteins are required for necroptosis. ( a ) Infection of L929 cells with ultraviolet-inactivated (UV) SeV rescues cell death as monitored by CellTiter-Glo (experiment repeated three times). Right panel: western blot of L929 cells infected with live or UV-inactivated SeV (10 HA U/ml) after 18 h (performed once) (41). ( b ) Schematic representation of SeV proteins. Six structural proteins (NP, P, M, F, HN, L) and accessory proteins (V, W, X, C, C', Y1, Y2). ( c ) L929 cells stably expressing a control (Ctl) shRNA or shRNA against P and accessory proteins (PVC) were treated with zVAD and infected with SeV (10 HA U/ml) and cell survival monitored. Right panel: knockdown was monitored by western blot using antibodies against the C proteins of SeV. ( d ) L929 cells were infected with Cantell strain of SeV, Z strain of SeV (SeV-Z), or mutants (d2Y-Z, V[-]-Z). 16–18 h post-infection, mRNA was isolated and used for quantitative RT-PCR using oligonucleotides specific for the SeV NP or γ -actin gene. Expression was plotted on a log scale relative to mock-infected samples after normalization to γ -actin. Standard error of the mean was calculated from three independent experiments. ( e ) Survival of cells infected with Cantell strain of SeV (SeV) or Z strain (SeV-Z, d2Y-Z, V[-]-Z) as measured by CellTiter-Glo assay. ( f ) Lysates from SeV-infected cells at various time points (M: mock-infected) were probed in a western blot analysis using SeV C protein or β-actin specific antibodies (experiment repeated twice). For 3A, 3C, 3D, 3E, data are averages of triplicates from a single experiment, which is representative of at least three independent experiments. *** P
Article Snippet: 16–18 hours post-infection, cell survival was measured by CellTiter-Glo ATP Assay (Promega).
Techniques: Infection, Western Blot, Stable Transfection, Expressing, CTL Assay, shRNA, Isolation, Quantitative RT-PCR, Glo Assay