(A) TNFα protein expression level in IPNV-infected ZF4 cells (MOI = 1) 0, 6, 12, and 24 h p.i. The protein was detected using western blot with a polyclonal antibody specific for TNFα. Lanes 1–4: ZF4 cells were pretreated with 50 μM AG-126 and infected with IPNV for 0 (lane 1), 6 (lane 2), 12 (lane 3), or 24 (lane 4). Lanes 5–7: Untreated ZF4 cells were infected with IPNV for 6 (lane 5), 12 (lane 6), or 24 h (lane 7). The expression of actin was used as an internal control. (B) TNFα mRNA expression in IPNV-infected ZF4 cells was quantified using RT-PCR. The ZF4 cells were pre-treated with 50 μM or 100 μM AG-126 for 2 hours, infected with IPNV (MOI = 1), and incubated for 0, 3, 6, 12, or 24 h. The expression of ef1a (elongation factor 1-alpha) was used as an internal control. (C) The tnfa expression was inhibited by TNFα-specific siRNA in IPNV-infected cells. TNFα expression was efficiently inhibited by TNFα-specific siRNA after IPNV infection. Sample 1: ZF4 cells infected by IPNV. Sample 2: ZF4 cells pretreated with scrambled siRNA and then infected by IPNV. Sample 3: ZF4 cells pretreated with TNFα-specific siRNA and then infected by IPNV. The quantification of gene expression in normal versus siRNA-treated cells was calculated relative to ef1a. (D) Detection of TNFα in untreated or TNFα-specific siRNA-treated ZF4 cells after IPNV infection by western blotting. Lane 1: untreated ZF4 cells; lane 2: ZF4 cells treated with control siRNA; lane 3: ZF4 cells treated with TNFα-specific siRNA. The expression of actin was used as an internal control. (E) Cell viability of IPNV-infected ZF4 cells pre-treated with TNFα-specific siRNA or AG-126 at 0, 6, 12, 24, 36 and 48 h p.i. The viability of each sample was determined in three individual experiments. Data shown are the mean ± SD. Student′s t tests indicate significant differences compared to IPNV infection only or untreated control: *, p<0.05; **, p<0.01.

(A) The expression level of the pro-apoptotic proteins Bad and Bid in IPNV-infected ZF4 cells (MOI = 1) at 0, 6, 12, and 24 h p.i. was determined. The proteins were detected using western blot with a polyclonal antibody specific for mouse Bad. Lanes 1–4: ZF4 cells were pretreated with TNFα-specific siRNA and infected with IPNV for 0 (lane 1), 6 (lane 2), 12 (lane 3), or 24 h (lane 4). Lanes 5–7: untreated ZF4 cells were infected with IPNV for 6 (lane 5), 12 (lane 6), or 24 h (lane 7). The expression of actin was used as an internal control. Results are expressed as the ratio of Bad/actin. The mRNA expression of bad (B) and bid (D) in IPNV-infected ZF4 cells was quantified using quantitative RT-PCR. ZF4 cells were pre-treated with TNFα-specific siRNA or AG-126 and infected with IPNV (MOI = 1) for 0, 6, 12, or 24 h. The expression of ef1a was used as an internal control. Data shown are mean ± SD. Student′s t tests indicate significant differences compared to untreated control: **, p<0.01. (C) The expression level of the pro-apoptotic proteins Bid and t-Bid in IPNV-infected ZF4 cells (MOI = 1) at 0, 6 and 12 h p.i. was determined. The proteins were detected using western blot with a polyclonal antibody specific for Bid. Lanes 2–3: ZF4 cells were pretreated with TNFα-specific siRNA and infected with IPNV for 6 (lane 2) or 12 h (lane 3). Untreated ZF4 cells were infected with IPNV for 6 (lane 4) or 12 h (lane 5). Untreated ZF4 cells were infected with IPNV for 0 h (Lane 1). The expression of actin was used as an internal control. Results are expressed as the ratio of Bid/actin or t-Bid/actin.

(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.