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Fig. 5 (A) Detection of ROS production in TNFα-specific siRNA or AG-126 pre-treated cells after IPNV infection at 0, 6, 12, 18 or 24 h p.i. Fluorescence assays were performed in triplicate. Determination of the percentage of PI-positive cells after IPNV infection. (B) Detection of RIP1 in untreated or TNFα-specific siRNA-treated ZF4 cells by western blotting. Lane 1: untreated IPNV-infected ZF4 cells; lane 2: IPNV-infected ZF4 cells treated with scrambled siRNA; lane 3: IPNV-infected ZF4 cells treated with RIP1-specific siRNA. The expression of actin was used as an internal control. Detection of annexin V-positive cells following infection with IPNV. ZF4 cells were pre-treated with RIP1-specific siRNA (C), Nec-1 (D), DPI (E) or BHA (F), infected with IPNV (MOI = 1), and incubated for 0, 6, 12, 18 and 24 h. Three individual experiments were performed for each sample. Data shown are the mean ± SD. Student′s t tests indicate significant differences compared to IPNV infection only: *, p<0.05.