Figure 4. PEDV-N promotes the acetylation/release of HMGB1 and expression of proinflammatory cytokines.

The protein concentration in the supernatant was determined using the Bradford assay. An equal amount of protein was used for western blot analysis. A. Vero cells were infected with UV-inactivated PEDV (UV-PEDV) at different MOI for 1h at 37°C. Total and acetylated HMGB1 in the supernatant were then immediately analyzed by western blot. B. Vero cells were infected with PEDV (MOI=0.1). PEDV-N in cells was analyzed by western blot at different time points of the infection. C. Vero cells were transfected with PCA-N or PCA (2.5μg) for 12h, 24h, 36h. The mRNA level of HMGB1 or PEDV-N was analyzed by qRT-PCR. D. Vero cells were transfected with PCA-N or PCA at different doses (0.5, 1μg) for 36h. The mRNA of HMGB1 or N was analyzed. E. and F. The acetylated and total HMGB1 in the supernatant were determined by western blot in the cells transfected with PCA-N or PCA at the different amount or time points. The protein concentration in the supernatant was determined using the Bradford assay. An equal amount of protein was used for western blot analysis. G. to I. Vero cells were first transfected with PCA-N or PCA at the same doses (2.5μg) for 12h and then treated with GAR, RES, or BAY for 24h. The acetylated HMGB1 and total HMGB1 in the supernatant were determined by western blot. J. Vero cells were transfected with PCA-N or PCA at the same doses (2.5μg) for 12h, 24h, 36h. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. K. Vero cells were transfected with PCA-N or PCA at the same doses (2.5μg) for 12h. The cells were incubated with 1.5μg HMGB1 antibody for 24h. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P < 0.05; **, P < 0.01.